Systems, Methods, And Compositions For Imaging Androgen Receptor Axis Activity In Carcinoma, And Related Therapeutic Compositions And Methods Patent Application (2024)

U.S. patent application number 15/777319 was filed with the patent office on 2018-11-15 for systems, methods, and compositions for imaging androgen receptor axis activity in carcinoma, and related therapeutic compositions and methods.The applicant listed for this patent is Memorial Sloan Kettering Cancer Center. Invention is credited to David Ulmert.

SYSTEMS, METHODS, AND COMPOSITIONS FOR IMAGING ANDROGEN RECEPTORAXIS ACTIVITY IN CARCINOMA, AND RELATED THERAPEUTIC COMPOSITIONSAND METHODS

Abstract

Presented herein are systems, compositions, and methods forimmuno-PET/SPECT and/or immuno-fluorescence-guided imaging oftissue for diagnosing, localizing, radiation dose planning, and/orevaluating therapy response (e.g., anti-androgen receptortherapeutics, surgery and external irradiation) in cancer (e.g.,androgen receptor (AR) positive breast cancer). In otherembodiments, for example, the invention is directed toradio-immunotherapy (RIT) treatment of AR-positive breast cancer byadministration (e.g., injection) of a free-PSA and/or free hK2antibody labelled with a radioisotope after KLK2 and KLK3 inductionby progesterone, testosterone, and/or irradiation.

Related U.S. Patent Documents

Goverment Interests

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grantnumbers CA096945, CA127768, CA092629, and CA008748 awarded by theNational Institutes of Health. The government has certain rights inthe invention.

Claims

1. A method of assessing androgen receptor activity in a subject,the method comprising: administering, to the subject, atracer-labelled hK2-specific or PSA-specific antibody; anddetecting the presence of the labeled antibody in a tissue of thesubject.

2. The method of claim 1, wherein the tissue comprises breasttissue.

3. The method of claim 1 or 2, wherein the antibody comprises amurine or humanized antibody.

4. The method of any one of claims 1 to 3, wherein the antibodycomprises murine or humanized 11B6, and/or murine or humanized5A10.

5. The method of any one of the preceding claims, wherein thetracer comprises a radionuclide.

6. The method of claim 5, wherein the radionuclide is a memberselected from the group consisting of .sup.11C, .sup.64Cu,.sup.124I, .sup.111In, .sup.177Lu, .sup.15O, .sup.18F, .sup.68Ga,.sup.89Zr, and .sup.82Rb.

7. The method of any one of the preceding claims, comprisingadministering hu11B6 labeled with .sup.89Zr or administering.sup.89Zr-DFO-hu11B6.

8. The method of any one of the preceding claims, wherein detectingis performed via PET imaging, CT imaging, SPECT imaging, and/or invivo imaging.

9. The method of claim 8, comprising detecting the presence and/oractivity of the androgen receptor (AR) axis.

10. The method of any one of the preceding claims, comprisingdetecting the presence of the labeled antibody in the tissue at atime frame selected from the group consisting of at least 24 hoursafter administration of the labeled antibody to the subject, atleast 48 hours after administration of the labeled antibody to thesubject, at least 100 hours after administration of the labeledantibody to the subject, and at least 120 hours afteradministration of the labeled antibody to the subject.

11. The method of claim 10, wherein the labeled antibodyaccumulates and internalizes in tumor cells, thereby allowingvisualization/tracking over long periods of time.

12. The method of any one of the preceding claims, wherein thetissue has metastasized to bone.

13. The method of any one of the preceding claims, comprisingdetecting the presence of the labeled antibody in the tissue over aperiod of multiple time intervals.

14. The method of claim 13, wherein the detecting is for real-timemonitoring/visualization.

15. The method of claim 13 or 14, comprising detecting the presenceof the labeled antibody in the tissue at multiple times, includingat least one detection after a time selected from the groupconsisting of at least 24 hours following administration of thelabeled antibody, after at least 48 hours following administrationof the labeled antibody, after at least 100 hours followingadministration of the labeled antibody, and after at least 120hours following administration of the labeled antibody.

16. The method of any one of the preceding claims, furthercomprising one or more of (i) to (vi), as follows: (i) identifyingthe presence of cancer in the subject; (ii) localizing a cancer inthe subject; (iii) quantitatively assessing androgen receptorpathway activity in the subject/cancer; (iv) planning radiationdose(s) in a course of treatment of the subject; (v) determiningone or more pharmacodynamics parameters for the subject; and (vi)evaluating treatment efficacy.

17. The method of claim 16, wherein the cancer comprises a memberselected from the group consisting of breast cancer (BCa),AR-positive breast cancer, triple negative breast cancer (TN-BCa),and any metastasis of BCa. Ar-positive breast cancer, andTN-BCa.

18. The method of claim 16 or 17, wherein the determining of one ormore pharmacodynamics parameters for the subject is in conjunctionwith treatment of the subject with one or more drugs.

19. The method of any one of claims 16 to 18, wherein theevaluating comprises evaluating therapy response.

20. The method of any one of claims 16 to 19, comprising monitoringAR-upregulation of KLK2 and/or KLK3.

21. The method of claim 20, wherein the AR-upregulation of KLK2and/or KLK3 is in response to external irradiation.

22. A method of assessing androgen receptor activity in a subject,the method comprising: administering, to the subject, atracer-labelled hK2-specific or PSA-specific antibody; anddetecting the presence of the labeled 11B6 in a tissue of thesubject.

23. The method of claim 22, wherein the tissue comprises breasttissue.

24. The method of claim 22 or 23, wherein the tracer-labelledhK2-specific or PSA-specific antibody comprises a murine orhumanized antibody.

25. The method of claim 24, wherein the murine or humanizedantibody comprises a murine or humanized 11B6 (hu11B6), and/ormurine or humanized 5A10 (hu5A10).

26. The method of any one of claims 22 to 25, wherein the tracercomprises a fluorophore.

27. The method of claim 26, comprising administering hu11B6 labeledwith a tag comprising a member selected from the group consistingof a near infrared fluorophore and a Cy5.5.

28. The method of any one of claims 22 to 27, wherein the detectingis performed via fluorescent imaging or in vivo imaging.

29. The method of any one of claims 22 to 28, comprising detectingthe presence and/or activity of the androgen receptor (AR)axis.

30. The method of any one of claims 22 to 29, further comprisingone or more of (i) to (vi), as follows: (i) identifying thepresence of cancer in the subject; (ii) localizing the cancer inthe subject; (iii) quantitatively assessing androgen receptorpathway activity in the subject/cancer; (iv) planning radiationdose(s) in a course of treatment of the subject; (v) determiningone or more pharmacodynamics parameters for the subject; and (vi)evaluating treatment efficacy.

31. The method of claim 30, wherein the cancer comprises a memberselected from the group consisting of breast cancer (BCa),AR-positive breast cancer, triple negative breast cancer (TN-BCa),and any metastasis of BCa, AR-positive breast cancer, andTN-BCa.

32. The method of claim 30 or 31, wherein the determining of one ormore pharmacodynamics parameters for the subject is determined inconjunction with treatment of the subject with one or moredrugs.

33. The method of any one of claims 30 to 32, comprising monitoringAR-upregulation of KLK2 and/or KLK3.

34. The method of claim 33, wherein the AR-upregulation of KLK2and/or KLK3 is in response to external irradiation.

35. A method of treating AR-positive breast cancer with one or moreagents/treatments selected from the group consisting of: (i) aradionuclide-labelled hK2-specific or PSA-specific antibody; and(ii) at least one member selected from the group consisting ofprogesterone, testosterone, and external irradiation, which methodcomprises administering the one or more agents/treatments to asubject suffering from or susceptible to AR-positive breast cancer,so that the subject is receiving therapy with a combination of (i)and (ii) above.

36. The method of claim 35, wherein the radionuclide comprises amember selected from the group consisting of .sup.90Y, .sup.131I,.sup.211At, .sup.111In, .sup.177Lu, .sup.227Th, .sup.149Tb,.sup.212Bi, .sup.213Bi, .sup.225Ac, .sup.82Rb, and .sup.223Ra.

37. The method of claim 35 or 36, wherein the radionuclide-labelledhK2-specific or PSA-specific antibody comprises a member selectedfrom the group consisting of a humanized 11B6 (hu11B6), humanized5A10 (hu5A10), hu11B6 labeled with an alpha-particle-emittingradionuclide, hu11B6 labeled with .sup.225Ac, and.sup.225Ac-DOTA-hu11B6.

38. A method of treating AR-positive breast cancer or anymetastasis of AR-positive breast cancer, the method comprisingadministering, to a subject suffering from or susceptible to thedisease or condition, a radionuclide-labelled hK2-specific orPSA-specific antibody.

39. The method of claim 38, wherein the radionuclide comprises amember selected from the group consisting of .sup.90Y, .sup.131I,.sup.211At, .sup.149Tb, .sup.212Bi, .sup.213Bi, .sup.225Ac,.sup.111In, .sup.177Lu, .sup.227Th, and .sup.223Ra.

40. A composition comprising one or more agents selected from thegroup consisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in a method of treating AR-positive breastcancer in a subject suffering from or susceptible to AR-positivebreast cancer, wherein the treating comprises: delivering acombination of (i) and (ii) above to the subject.

41. A composition comprising one or more agents selected from thegroup consisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in therapy.

42. A composition comprising one or more agents selected from thegroup consisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in a method of in vivo diagnosis ofAR-positive breast cancer in a subject in a subject suffering fromor susceptible to AR-positive breast cancer, wherein the in vivodiagnosis comprises: delivering a combination of (i) and (ii) aboveto the subject.

43. A composition comprising one or more agents selected from thegroup consisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in in vivo diagnosis.

44. A composition comprising one or more agents selected from thegroup consisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in (a) a method of treating AR-positive breastcancer in a subject or (b) a method of in vivo diagnosis ofAR-positive breast cancer in a subject, wherein the methodcomprises: delivering a combination of (i) and (ii) above to thesubject.

45. The composition of any one of claims 40 to 44, wherein theradionuclide comprises a member selected from the group consistingof .sup.90Y, .sup.131I, .sup.211At, .sup.111In, .sup.177Lu,.sup.227Th, .sup.149Tb, .sup.212Bi, .sup.213Bi, .sup.225Ac,.sup.82Rb, and .sup.223Ra.

46. The composition of any one of claims 40 to 45, wherein theradionuclide-labelled hK2-specific or PSA-specific antibodycomprises a member selected from the group consisting of ahumanized 11B6 (hu11B6), humanized 5A10 (hu5A10), hu11B6 labeledwith an alpha-particle-emitting radionuclide, hu11B6 labeled with.sup.225Ac, and .sup.225Ac-DOTA-hu11B6.

47. A composition comprising a radionuclide-labelled hK2-specificof PSA-specific antibody for use in a method of treatingAR-positive breast cancer or any metastasis of AR-positive breastcancer in a subject suffering from or susceptible to the disease orcondition, wherein the treating comprises delivering thecomposition to the subject.

48. A composition comprising a radionuclide-labelled hK2-specificof PSA-specific antibody for use in therapy.

49. A composition comprising a radionuclide-labelled hK2-specificof PSA-specific antibody for use in a method of in vivo diagnosisof AR-positive breast cancer or any metastasis of AR-positivebreast cancer in a subject suffering from or susceptible to thedisease or condition, wherein the in vivo diagnosis comprisesdelivering the composition to the subject.

50. A composition comprising a radionuclide-labelled hK2-specificof PSA-specific antibody for use in in vivo diagnosis.

51. A composition comprising a radionuclide-labelled hK2-specificof PSA-specific antibody for use in (a) a method of treatingAR-positive breast cancer or any metastasis of AR-positive breastcancer in a subject or (b) a method of in vivo diagnosis ofAR-positive breast cancer or any metastasis of AR-positive breastcancer in a subject, wherein the method comprises delivering thecomposition to the subject.

52. The composition of claim 47 or 51, wherein the radionuclidecomprises a member selected from the group consisting of .sup.90Y,.sup.131I, .sup.211At, .sup.149Tb, .sup.212Bi, .sup.213Bi,.sup.225Ac, .sup.111In, .sup.177Lu, .sup.223Ra, and .sup.227Th.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 62/257,179, filed Nov. 18, 2015, the contents ofwhich is hereby incorporated by reference herein in itsentirety.

TECHNICAL FIELD

[0003] Presented herein are systems, methods, and compositions forimaging, diagnosing, and/or treating cancer, for example, androgenreceptor positive breast cancer.

BACKGROUND

[0004] Activation of the androgen receptor (AR) signaling axiscontributes to prostate cancer (PCa) progression throughout theentire course of the disease, including the castration resistantstate. Following initial response to inhibition with androgendeprivation treatment, a mainstay of PCa treatment, AR-pathwayreactivation inevitably occurs. Reactivation has been attributed togene amplification, intratumoral androgen synthesis, constitutivelyactive AR variants, and other mechanisms. AR is also differentiallyexpressed in several breast cancer (BCa) subtypes, though without aclearly defined role. This includes aggressive triple negative BCa(TN-BCa), where AR expression is correlated with decreasedsurvival. Recent trials have focused on AR inhibition as anapproach to stabilize this otherwise unmanageable disease. Thus,quantifying lesion-specific AR pathway activity represents acritical unmet need that assists in treatment selection, as apharmacodynamic marker of pathway inhibition, and represents anon-invasive biomarker of therapeutic efficacy.

[0005] Prostate specific antigen (PSA), also known as kallikrein-3(KLK3), is a commonly used biomarker of prostate cancer. AlthoughPSA is androgen regulated, concentrations in the blood are afunction of degree of tumor differentiation, physiological factorsand total tumor burden, making PSA unsuitable as a measure ofpathway activation.

[0006] In humans, KLK2 is a gene that encodes kallikrein relatedpeptidase 2 (hK2), a trypsin-like enzyme with AR-driven expressionspecific to prostate tissue, PCa and AR-positive BCa tissues. hK2is activated by Transmembrane Protease, Serine 2 (TMPRSS2) andsecreted into the ducts of the prostate, where it initiates acascade that cleaves sem*nogelin, the extracellular matrix inejacul*te, to enhance sperm motility. hK2 in man is exclusivelyexpressed in prostatic tissues (FIG. 9D). Similar to PSA,retrograde release of catalytically inactive hK2 into the bloodoccurs when the highly structured organization of the prostate iscompromised upon hypertrophy or malignant transformation.

[0007] The AR axis is active in many difficult-to-treat breastcancer (BCa) subtypes, such as triple negative breast cancer(TN-BCa), and in treatment-resistant disease (e.g., anti-estrogentherapy or tamoxifen resistance). KLK2 expression is low in BCacells, but can be increased by treatment with progesterone,testosterone, and/or external irradiation.

[0008] Inhibition of disease processes by drug binding to secretedantigens is established in clinical practice. Targets of biologicsinclude vascular endothelial growth factor, receptor activator ofnuclear factor kappa-B and tissue necrosis factor, among others.Imaging agents or drug conjugates directed to secreted antigenshave been far less successful, as antibody-bound complexes wash outof the disease site. This has limited targets for PCa to cellsurface receptors, which usually have poor tissue- ordisease-restricted expression (FIGS. 9A-9C), as indicated from theintegrated in silico transcriptomics database (IST,Medisapiens).

[0009] Direct imaging of AR abundance and measurement of receptoroccupancy has previously been achieved using .sup.18F-FDHT (aradiolabeled analog of the androgen testosterone). However, uptakeof this agent does not correlate with PSA decline or response, bothof which are positively tied to AR pathway activity and not simplythe amount of receptor. In the VCaP prostate cancer model, therapid metabolism and abdominal clearance of this agent (FIG. 28)results in limited contrast of tumor to background structures.

[0010] Therefore, there is a need for improved systems, methods,and compositions to characterize disease, guide therapy, andmonitor response of treatment.

SUMMARY

[0011] Presented herein are systems, compositions, and methodsinvolving the use of murine and/or humanized antibodies targetingfree PSA (such as 5A10) and/or free hK2 (such as 11B6) for in vivotargeting of androgen receptor (AR) positive cancer (e.g., breastcancer, e.g., prostate cancer). For example, the antibodies can beused alone (e.g., 5A10 or 11B6) or in combination (e.g., 5A10 and11B6).

[0012] For example, in certain embodiments, the present disclosureis directed to immuno-PET/SPECT and/or immuno-fluoresce-guidedimaging for diagnosing, localizing, radiation dose planning, and/orevaluating therapy response (e.g., anti-androgen receptortherapeutics, surgery and external irradiation) in androgenreceptor (AR) positive breast cancer or PCa. Evaluation can includemonitoring of AR-upregulation of KLK2 and KLK3 in response toexternal irradiation.

[0013] In other embodiments, for example, the present disclosure isdirected to radio-immunotherapy (RIT) treatment of AR-positivebreast cancer by administration (e.g., injection) of a free-PSAand/or free hK2 antibody labelled with a radioisotope after KLK2and KLK3 induction by progesterone, testosterone orirradiation.

[0014] In other embodiments, for example, the present disclosureprovides an antibody-based platform directed to a secreted antigenthat uses Fc-receptor mediated internalization for cancer imagingand therapy.

[0015] In one aspect, the invention is directed to a method ofassessing androgen receptor activity in a subject, the methodcomprising: administering, to the subject, a tracer-labelledhK2-specific or PSA-specific antibody; and detecting the presenceof the labeled antibody in a tissue of the subject.

[0016] In certain embodiments, the tissue comprises breasttissue.

[0017] In certain embodiments, the antibody comprises a murine orhumanized antibody. In certain embodiments, the antibody comprisesmurine or humanized 11B6, and/or murine or humanized 5A10.

[0018] In certain embodiments, the tracer comprises a radionuclide.In certain embodiments, the radionuclide is a member selected fromthe group consisting of .sup.11C, .sup.64Cu, .sup.124I, .sup.111In,.sup.177Lu, .sup.15O, .sup.18F, .sup.68Ga, .sup.89Zr, and.sup.82Rb.

[0019] In certain embodiments, the method comprises administeringhu11B6 labeled with .sup.89Zr or administering.sup.89Zr-DFO-hu11B6.

[0020] In certain embodiments, the detecting is performed via PETimaging, CT imaging, SPECT imaging, and/or in vivo imaging. Incertain embodiments, the method comprises detecting the presenceand/or activity of the androgen receptor (AR) axis. In certainembodiments, the method comprises detecting the presence of thelabeled antibody in the tissue at a time frame selected from thegroup consisting of at least 24 hours after administration of thelabeled antibody to the subject, at least 48 hours afteradministration of the labeled antibody to the subject, at least 100hours after administration of the labeled antibody to the subject,and at least 120 hours after administration of the labeled antibodyto the subject.

[0021] In certain embodiments, the labeled antibody accumulates andinternalizes in tumor cells, thereby allowingvisualization/tracking over long periods of time.

[0022] In certain embodiments, the tissue has metastasized tobone.

[0023] In certain embodiments, the method comprises detecting thepresence of the labeled antibody in the tissue over a period ofmultiple time intervals. In certain embodiments, the detecting isfor real-time monitoring/visualization.

[0024] In certain embodiments, the method comprises detecting thepresence of the labeled antibody in the tissue at multiple times,including at least one detection after a time selected from thegroup consisting of at least 24 hours following administration ofthe labeled antibody, after at least 48 hours followingadministration of the labeled antibody, after at least 100 hoursfollowing administration of the labeled antibody, and after atleast 120 hours following administration of the labeledantibody.

[0025] In certain embodiments, the method further comprises one ormore of (i) to (vi), as follows: (i) identifying the presence ofcancer in the subject; (ii) localizing a cancer in the subject;(iii) quantitatively assessing androgen receptor pathway activityin the subject/cancer; (iv) planning radiation dose(s) in a courseof treatment of the subject; (v) determining one or morepharmacodynamics parameters for the subject; and (vi) evaluatingtreatment efficacy. In certain embodiments, the cancer comprises amember selected from the group consisting of breast cancer (BCa),AR-positive breast cancer, triple negative breast cancer (TN-BCa),and any metastasis of BCa. Ar-positive breast cancer, andTN-BCa.

[0026] In certain embodiments, the determining of one or morepharmacodynamics parameters for the subject is in conjunction withtreatment of the subject with one or more drugs.

[0027] In certain embodiments, the evaluating comprises evaluatingtherapy response.

[0028] In certain embodiments, the method comprises monitoringAR-upregulation of KLK2 and/or KLK3. In certain embodiments, theAR-upregulation of KLK2 and/or KLK3 is in response to externalirradiation.

[0029] In another aspect, the invention is directed to a method ofassessing androgen receptor activity in a subject, the methodcomprising: administering, to the subject, a tracer-labelledhK2-specific or PSA-specific antibody; and detecting the presenceof the labeled 11B6 in a tissue of the subject.

[0030] In certain embodiments, the tissue comprises breasttissue.

[0031] In certain embodiments, the tracer-labelled hK2-specific orPSA-specific antibody comprises a murine or humanized antibody. Incertain embodiments, the murine or humanized antibody comprises amurine or humanized 11B6 (hu11B6), and/or murine or humanized 5A10(hu5A10).

[0032] In certain embodiments, the tracer comprises afluorophore.

[0033] In certain embodiments, the method comprises administeringhu11B6 labeled with a tag comprising a member selected from thegroup consisting of a near infrared fluorophore and a Cy5.5.

[0034] In certain embodiments, the detecting is performed viafluorescent imaging or in vivo imaging. In certain embodiments, themethod comprises detecting the presence and/or activity of theandrogen receptor (AR) axis.

[0035] In certain embodiments, the method further comprises one ormore of (i) to (vi), as follows: (i) identifying the presence ofcancer in the subject; (ii) localizing the cancer in the subject;(iii) quantitatively assessing androgen receptor pathway activityin the subject/cancer; (iv) planning radiation dose(s) in a courseof treatment of the subject; (v) determining one or morepharmacodynamics parameters for the subject; and (vi) evaluatingtreatment efficacy.

[0036] In certain embodiments, the cancer comprises a memberselected from the group consisting of breast cancer (BCa),AR-positive breast cancer, triple negative breast cancer (TN-BCa),and any metastasis of BCa, AR-positive breast cancer, andTN-BCa.

[0037] In certain embodiments, the determining of one or morepharmacodynamics parameters for the subject is determined inconjunction with treatment of the subject with one or moredrugs.

[0038] In certain embodiments, the method comprises monitoringAR-upregulation of KLK2 and/or KLK3. In certain embodiments, theAR-upregulation of KLK2 and/or KLK3 is in response to externalirradiation.

[0039] In another aspect, the invention is directed to a method oftreating AR-positive breast cancer with one or moreagents/treatments selected from the group consisting of: (i) aradionuclide-labelled hK2-specific or PSA-specific antibody; and(ii) at least one member selected from the group consisting ofprogesterone, testosterone, and external irradiation, which methodcomprises administering the one or more agents/treatments to asubject suffering from or susceptible to AR-positive breast cancer,so that the subject is receiving therapy with a combination of (i)and (ii) above.

[0040] In certain embodiments, the radionuclide comprises a memberselected from the group consisting of .sup.90Y, .sup.131I,.sup.211At, .sup.111In, .sup.177Lu, .sup.227Th, .sup.149Tb,.sup.212Bi, .sup.213Bi, .sup.225Ac, .sup.82Rb, and .sup.223Ra. Incertain embodiments, the radionuclide-labelled hK2-specific orPSA-specific antibody comprises a member selected from the groupconsisting of a humanized 11B6 (hu11B6), humanized 5A10 (hu5A10),hu11B6 labeled with an alpha-particle-emitting radionuclide, hu11B6labeled with .sup.225Ac, and .sup.225Ac-DOTA-hu11B6.

[0041] In another aspect, the invention is directed to a method oftreating AR-positive breast cancer or any metastasis of AR-positivebreast cancer, the method comprising administering, to a subjectsuffering from or susceptible to the disease or condition, aradionuclide-labelled hK2-specific or PSA-specific antibody.

[0042] In certain embodiments, the radionuclide comprises a memberselected from the group consisting of .sup.90Y, .sup.131I,.sup.211At, .sup.149Tb, .sup.212Bi, .sup.213Bi, .sup.225Ac,.sup.111In, .sup.177Lu, .sup.227Th, and .sup.223Ra.

[0043] In another aspect, the invention is directed to acomposition comprising one or more agents selected from the groupconsisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in a method of treating AR-positive breastcancer in a subject suffering from or susceptible to AR-positivebreast cancer, wherein the treating comprises: delivering acombination of (i) and (ii) above to the subject.

[0044] In another aspect, the invention is directed to acomposition comprising one or more agents selected from the groupconsisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in therapy.

[0045] In another aspect, the invention is directed to acomposition comprising one or more agents selected from the groupconsisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in a method of in vivo diagnosis ofAR-positive breast cancer in a subject in a subject suffering fromor susceptible to AR-positive breast cancer, wherein the in vivodiagnosis comprises: delivering a combination of (i) and (ii) aboveto the subject.

[0046] In another aspect, the invention is directed to acomposition comprising one or more agents selected from the groupconsisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in in vivo diagnosis.

[0047] In another aspect, the invention is directed to acomposition comprising one or more agents selected from the groupconsisting of: (i) a radionuclide-labelled hK2-specific orPSA-specific antibody; and (ii) at least one member selected fromthe group consisting of progesterone, testosterone, and externalirradiation, for use in (a) a method of treating AR-positive breastcancer in a subject or (b) a method of in vivo diagnosis ofAR-positive breast cancer in a subject, wherein the methodcomprises: delivering a combination of (i) and (ii) above to thesubject.

[0048] In certain embodiments, the radionuclide comprises a memberselected from the group consisting of .sup.90Y, .sup.131I,.sup.211At, .sup.111In, .sup.177Lu, .sup.227Th, .sup.149Tb,.sup.212Bi, .sup.213Bi, .sup.225Ac, .sup.82Rb, and .sup.223Ra. Incertain embodiments, the radionuclide-labelled hK2-specific orPSA-specific antibody comprises a member selected from the groupconsisting of a humanized 11B6 (hu11B6), humanized 5A10 (hu5A10),hu11B6 labeled with an alpha-particle-emitting radionuclide, hu11B6labeled with .sup.225Ac, and .sup.225Ac-DOTA-hu11B6.

[0049] In another aspect, the invention is directed to acomposition comprising a radionuclide-labelled hK2-specific ofPSA-specific antibody for use in a method of treating AR-positivebreast cancer or any metastasis of AR-positive breast cancer in asubject suffering from or susceptible to the disease or condition,wherein the treating comprises delivering the composition to thesubject.

[0050] In another aspect, the invention is directed to acomposition comprising a radionuclide-labelled hK2-specific ofPSA-specific antibody for use in therapy.

[0051] In another aspect, the invention is directed to acomposition comprising a radionuclide-labelled hK2-specific ofPSA-specific antibody for use in a method of in vivo diagnosis ofAR-positive breast cancer or any metastasis of AR-positive breastcancer in a subject suffering from or susceptible to the disease orcondition, wherein the in vivo diagnosis comprises delivering thecomposition to the subject.

[0052] In another aspect, the invention is directed to acomposition comprising a radionuclide-labelled hK2-specific ofPSA-specific antibody for use in in vivo diagnosis.

[0053] In another aspect, the invention is directed to acomposition comprising a radionuclide-labelled hK2-specific ofPSA-specific antibody for use in (a) a method of treatingAR-positive breast cancer or any metastasis of AR-positive breastcancer in a subject or (b) a method of in vivo diagnosis ofAR-positive breast cancer or any metastasis of AR-positive breastcancer in a subject, wherein the method comprises delivering thecomposition to the subject.

[0054] In certain embodiments, the radionuclide comprises a memberselected from the group consisting of .sup.90Y, .sup.131I,.sup.211At, .sup.149Tb, .sup.212Bi, .sup.213Bi, .sup.225Ac,.sup.111In, .sup.177Lu, .sup.223Ra, and .sup.227Th.

[0055] The description of elements of one aspect of the invention(e.g., features of a system, method, or composition) can be appliedas elements of other aspects of the invention (e.g., features of asystem, method, and/or composition) as well.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIGS. 1A-1D show prostate cancer targeting and accumulationof Active-hK2 Targeted Radiolabeled Antibody.

[0057] FIG. 1A shows coronal slices through xenograft (LNCaP)bearing mice, over time. The long-lived PET isotope .sup.89Zrenables longitudinal imaging, which shows continued uptake over 10d. Schematic of tumor location Tumor (T) on flank, and Liver (L).B

[0058] FIGS. 1B and 1C show biodistribution of mass escalationstudy at 320 h, with time time activity curves in % IA/g of tumor(squares) and blood (circles) for 50, 150 and 300 .mu.g doses (topto bottom of FIG. 1C).

[0059] FIG. 1D shows greater uptake in the higher-hK2 producingVCaP in comparison to the LNCaP and non-producing DU145 xenograftsindicates specificity, which can also be blocked with cold antibody(1 mg).

[0060] FIGS. 2A-2C show that .sup.89Zr-DFO-11B6 delineatesosteolytic and osteoblastic bone metastases. The radiotracer isable to distinguish both LNCaP osteolytic (FIG. 1A), VCaPosteoblastic tumors (FIG. 1B), and PC3 AR- and hK2-negativeosteolytic lesions (FIG. 1C) in the mouse tibia. X-ray computedtomography of the electron dense bone (left-most column; CT) showsthe loss of bone in the LNCaP and PC3 models. The intensity ofsignal again recapitulates the relative expression levels of thetwo AR-positive cell lines (PET column). 3-dimensional PET/CTfusion images with opaque bone (second from right) and transparentbone (right-most column) show that these metastases are restrictedfrom the surrounding. Low-levels of nonspecific .sup.89Zr uptake atthe epiphyseal growth plate is seen in all models. Quantitation ofuptake and kinetics are shown in FIGS. 13A-13B.

[0061] FIGS. 3A-3G show intracellular accumulation of 11B6-hK2.

[0062] FIGS. 3A-3E show that the whole prostate and seminalvesicles (prostate package) were removed from Pb_KLK2 mice 72 hafter injection of Cy5.5-11B6 and .sup.89Zr-DFO-11B6 for wholemount fluorescence (FIG. 3A), confocal microscopy (FIG. 3B), andautoradiography (FIG. 3C). Intense uptake in the glandularstructures of the ventral prostate (arrow), with lower uptake inthe dorsolateral prostate (*). No uptake in non-transgenic mice wasobserved (not shown). Radio- and fluorescent signal correlated withthe ventral prostate gland by H&E (FIG. 3D), which is confirmedby Androgen Receptor (AR) staining that is intense in the ventralprostate (scale is 500 .mu.m) (FIG. 3E).

[0063] FIGS. 3F-3G show that following incubation with LNCaPprostate cancer cells, the 11B6 antibody co-localizes with FcRNearly (FIG. 3F) and is then trafficked to acidified lysosomes asindicated by increased fluorescence from pH-responsive dye labeled11B6 (pH-11B6) (FIG. 3G).

[0064] FIG. 4 shows noninvasive annotation of prostate cancerdevelopment by .sup.89Zr-11B6. Representative pelvic fused.sup.89Zr-11B6 (50 .mu.g) PET/CT acquisitions of cancer susceptiblehK2-expressing mice (Pb_KLK2.times.Hi-Myc mice) throughoutdevelopment of adenocarcinoma. The age in weeks is displayed withinsert.

[0065] FIGS. 5A-5F show lesion response to treatment.

[0066] FIG. 5A shows representative PET imaging with .sup.89Zr-11B6on an intra-osseous LNCaP-AR model before (left) and after (right)castration.

[0067] FIGS. 5B-5C show that quantification of imaging results of.sup.89Zr-11B6 radiotracer uptake reflects AR-driven luciferasesignal changes in the LNCaP-AR cell line.

[0068] FIG. 5D shows, in contrast to FIG. 5C, that PSA bloodconcentration values remained unchanged.

[0069] FIG. 5E shows that conventional .sup.18F--NaF imaging wasalso conducted before (left) and after castration (right) prior to.sup.89Zr-11B6.

[0070] FIG. 5F shows that quantitation of bone scan uptake valuesillustrates continued bone turnover at the site of the resolvedlesion. Imaging experiments (n=5) and PSA assay (n=4), per group(Table 6).

[0071] FIGS. 6A-6E show characterization of drug response tosurgical castration and adjuvant androgen receptor blockage.Noninvasive longitudinal quantification of castration andanti-androgen therapy with .sup.89Zr-11B6. Pb_KLK2.times.Hi-Mycmice were imaged before treatment, after castration (6 weekspost-surgery) and after adjuvant therapeutic intervention (4 weeksafter either vehicle or enzalutamide (ENZ). Two representativesubjects in both the vehicle (PBS) (FIG. 6A) and Enzalutamidetreatment-groups (FIG. 6B).

[0072] FIG. 6C shows quantification (mean % ID/g) enabled by.sup.89Zr-11B6 of uptake of all mice pre- and post-castration.

[0073] FIGS. 6D and 6E show the mean uptake in the Vehicle (n=6)(FIG. 6D) and Enzalutamide groups (n=4) (FIG. 6E) through theentire adjuvant treatment regimen. Reactivation in the castrationplus androgen receptor blockade group was not significant(n.s.).

[0074] FIGS. 7A and 7B show AR-increase after irradiation of twoAR-positive BCa cell lines (BT474 and MFM223).

[0075] FIG. 8 shows a survival graph after injecting.sup.225Ac-DOTA-hu11B6 in DHT-stimulated (e.g., expression of KLK2)and in non-DHT stimulated mice (e.g., non-KLK2 expression).

[0076] FIGS. 9A-9D show anatomical and disease-specific geneexpression of candidate targets. Targeted agents for diseaseidentification, characterization, and therapy include FIG. 9A) sixtransmembrane epithelial antigen of the prostate 1 (STEAP1), (FIG.9B) prostate-specific membrane antigen (FOLH1), and (FIG. 9C) GCPRbombesin receptor (BSR3). FIG. 9D shows AR-activity regulated humankallikrein-related peptidase 2 (KLK2) is restricted to the prostateand prostate-derived tissue, as well as adenocarcinoma of thebreast under sex-steroid stimulation. Median expression is shown asa horizontal line, with 25 and 75 percentiles as lower and upperbounds of the boxes, with whiskers and outlier points extending tocover the remaining data. Data from the In Silico TranscriptomicsOnline database, an integrated human gene expression catalog of 60healthy tissues (light speckling), 104 malignant, and 64 otherdisease types (dark speckling).

[0077] FIG. 10 shows a competition assay comparing the affinity ofnon-labeled 11B6 (square) to DFO-conjugated (open circle), as wellas .sup.89Zr-labeled DFO-11B6 (closed circle). No significantdifferences in capture efficacy of free hK2 are noted for theconjugated or radiolabeled constructs.

[0078] FIGS. 11A and 11B show .sup.89Zr-DFO-11B6 uptake and hK2expression.

[0079] FIG. 11A shows protein expression and uptake of the tracerwere correlated. Percent injected activity values were assessed bygamma-counting, and hK2 from lysate was measured by time-resolvedimmunofluorometric assay. hK2 protein values are expressed as ngper mg of total protein.

[0080] FIG. 11B shows that implanted 22Rv1 xenografts into theflank of castrated Balb/c nu/nu mice was used to evaluate theuptake of the tracer in a model of patients who have failed hormonetherapy. Biodistribution demonstrates uptake at the tumor, throughcontinued AR-driven hK2 expression.

[0081] FIGS. 12A-12D show relative expression of putative prostatemarkers in prostate cancer cell lines. RT-PCR was performed on 7commonly used prostate cancer cell lines for genes of interestwhich included (FIG. 12A) KLK2 (encoding hK2), (FIG. 12B) FOLH1(encoding PSMA, prostate-specific membrane antigen), and (FIG. 12C)KLK3 (encoding PSA). FIG. 12D shows neonatal Fc Receptor GeneExpression encoding the IgG-binding neonatal Fc receptor, across apanel of prostate cancer cell lines.

[0082] FIGS. 13A and 13B show time-activity curves of LNCaP-ARsubcutaneous and orthotopic xenografts. FIG. 13A shows the kineticsof uptake measured as % IA/g in the flank xenograft model arefaster than in (FIG. 13B) an orthotopic bone model. Time-activitycurves were plotted noninvasively from dynamic PET acquisitions atthe times indicated and show tumor (square) and blood (circle)values. Blood values were assessed from the mean % IA/g of volumesof interest defined around the heart from PET datasets.

[0083] FIGS. 14A-14C show .sup.89Zr-DFO-11B6 prostate andhK2-specific imaging in transgenic healthy and diseased mice.Sagittal and oblique views of three-dimensional .sup.89Zr-11B6 (50.mu.g) PET/CT fusion volumes of the pelvis in representative mice,with surface-rendered skeleton, 96 h after administration.

[0084] FIG. 14A shows no uptake of the radiotracer is seen in awild-type C57Bl/6 mouse (42 weeks).

[0085] FIG. 14B shows a representative image of a mouse (51 weeks)that has been engineered to express the active hK2 protein under aprostate-specific promoter. At tracer dose, the 11B6 imaging agentis able to define the two ventral lobes (which express the mostprotein).

[0086] FIG. 14C shows that crossing these transgenic animals withestablished models of prostate cancer, for example, thisrepresentative hK2.times.Hi-Myc mouse, yields greater uptake of thetracer in the cancerous prostate. Note that intact antibodies areexcreted primarily through the liver, and therefore bladder signalis not expected or seen.

[0087] FIGS. 15A-15D show Cy5.5-11B6 cellular uptake.

[0088] FIG. 15A-15B show white light and fluorescence imaging of asingle cell suspension of hK2-expressing mouse prostate afterintravenously administered Cy5.5-11B6, respectively.

[0089] FIG. 15C shows confocal microscopy of cultured VCaP cellsincubated with Cy5.5-11B6 overlaid on differential interferencecontrast light image of cells.

[0090] FIG. 15D shows three dimensional rendering of fluorescencedistribution within the cells in XY (top) and YZ (bottom)perspectives.

[0091] FIGS. 16A and 16B show FcRn-specific transport.

[0092] FIG. 16A shows SPR determined dissociation constants forFcRn for 11B6 and H435A-11B6 at pH 6 and 7.4.

[0093] FIG. 16B shows exploiting the receptor's pH-dependentaffinity, FcRn-mediated uptake is confirmed by increased uptakekinetics at low extracellular pH. Uptake is abrogated withH435A-modified 11B6.

[0094] FIGS. 17A-17F show investigation of FcRn-mediated uptake of11B6 complexed with hK2.

[0095] FIG. 17A shows a comparison of uptake in LNCaP xenografts(and blood clearance from heart measurements) of the 11B6 antibody,and the single point mutated H435A-11B6.

[0096] FIG. 17B shows ex vivo organ and tumor biodistribution ofantibody uptake at 320 h.

[0097] FIG. 17C shows in vitro verification of binding of both 11B6and the H435A mutant to hK2 by immunofluorimetric competitionassay.

[0098] FIG. 17D shows validation of the uptake of the intactantibody (11B6) and the lack of uptake of the antibody with anFc-specific single amino acid point mutation (H435A) inhK2-expressing GEM (Pb_KLK2).

[0099] FIG. 17E shows ex vivo biodistribution of the twonon-accumulating constructs (non-specific IgG1 and H435A) thatdemonstrates a requirement for both hK2 binding and FcRninternalization.

[0100] FIG. 17F shows biodistribution data at 320 h of hu11B6 andH435A.

[0101] FIG. 18 shows uptake of pH-dye labeled 11B6. Top row: 11B6,bottom row: control IgG. Prostate cancer cells (LNCaP) were pulsedwith pH indicator dye-labeled antibody. Internalized 11B6 is not inan acidic environment at 12 h (but has been internalized; FIGS.3A-3G). Fluorescence intensity increased in the acidic lateendosomes at later time points. Control IgG was not detected.

[0102] FIGS. 19A-19G show imaging cross-activation of the ARpathway in LREX' models.

[0103] FIGS. 19A-19E show biodistribution of --Zr-DFO-11B6 in flankxenografts of the enzalutamide-resistant LREX' model in castratedanimals with daily enzalutamide and dexamethasone treatment. Amodel of LREX' liver metastasis was developed by orthotopicimplantation of the cells in Matrigel in animals similarlysupplemented with dexamethasone and enzalutamide. Metastasis burdenwas monitored by (FIG. 19B) bioluminescent imaging and (FIGS.19C-19E) PET/MR using .sup.89Zr-DFO-11B6.

[0104] FIGS. 19F and 19G show H&E and autoradiography of thedistribution of the tracer at metastatic deposits within theliver.

[0105] FIG. 20 shows accumulation of re-engineered anti-PSAantibody. Radiolabeled .sup.89Zr-antibody uptake in LNCaP flanktumors in nude mice. 5A10, an antibody targeting free PSA,experiences transient uptake in LNCaP xenografts (black, closedcircles). When the CDR binding regions were grafted onto the 11B6antibody scaffold and retaining free PSA specificity(5A10.sup.H435-wt, it was observed that non-transient tumoralaccumulation of the antibody (blue, open circles).

[0106] FIGS. 21A-21E show hK2 production after DHT stimulation.AR-positive breast cancer cell lines were found to secrete hK2 intothe cell culture medium as detected by immunofluorimetric assayafter DHT stimulation. The values for free hK2 (fhK2) for thepositive cell lines (FIG. 21A) BT-474 and (FIG. 21B) MFM-223 areshown here without treatment (vehicle; VEH), with irrelevanthormone addition (estrogen; EST), and with testosterone (DHT). Notethat the plots are on a log 10 scale. RT-PCR was performed on thecells to compare the expression of KLK2 and FOLH1 in (FIG. 21C)BT-474 and (FIG. 21D) MFM-223. FIG. 21E shows in vivobiodistribution of .sup.89Zr-11B6 in BT474 xenografts with estrogenand DHT stimulation.

[0107] FIG. 22 shows a PET/CT image of .sup.89Zr-DFO-11B6 in asubcutaneous MFM223 model following DHT stimulation, revealing thepresence of AR+ triple negative breast cancer.

[0108] FIG. 23 shows intracellular accumulation of 11B6-hK2 inbreast cancer cells. Under DHT stimulation, the AR-positive BT474expresses hK2. Conjugated 11B6 is internalized in a time-dependentmanner by the stimulated cells. Cy5.5-11B6, red; DAPI, blue.

[0109] FIGS. 24A-24E show quantitation of .sup.89Zr-11B6 uptake intransgenic PCa mice.

[0110] FIG. 24A shows .sup.89Zr-DFO-11B6 uptake in the transformedprostate was determined non-invasively by volume of interestmeasurement at baseline (ages 18-24 weeks).

[0111] FIGS. 24B-24D show ex vivo autoradiography and histologyconfirm prostate and tumor specific uptake.

[0112] FIG. 24E shows quantification of PET before and aftercastration.

[0113] FIGS. 25A-25E show serial PET/CT monitoring .sup.89Zr-11B6uptake before, during, and after reversible castration by GNRHreceptor blockade.

[0114] FIG. 25A shows treatment and .sup.89Zr-11B6 PET imagingschedule throughout testosterone-depleting degarelix therapy.

[0115] FIG. 25B shows initial PET/CT prior to treatment (12 weeksof therapy consisting of 2 consecutive depot injections ofdegarelix acetate).

[0116] FIGS. 25C-25E show representative images 2, 10, and 14 weeksafter treatment initiation, respectively.

[0117] FIGS. 26A-26C show noninvasive monitoring of AR status with.sup.89Zr-DFO-11B6.

[0118] FIG. 26A shows relative expression of KLK2 in prostatetissue collected from Pb_KLK2 XHi-Myc mice without treatment, withcastration and vehicle (saline) and with castration and adjuvantenzalutamide.

[0119] FIG. 26B shows PCR analysis of KLK2 gene expression intissues treated with castration and enzalutamide resected by.sup.89Zr-DFO-11B6 guidance (shaded) and prostate tissues negativefor 11B6 uptake (white).

[0120] FIG. 26C shows a plot of the amount of hK2 protein(normalized to the total protein concentration) of tissues fromPb_KLK2 XHi-Myc treated with full androgen blockade correlated with.sup.89Zr-DFO-11B6 uptake minimum (blue) and maximum (red) values.The plot shows that uptake quantified by PET correlates to theactual hK2 protein level.

[0121] FIGS. 27A-27N show multimodality imaging for pre- andintra-operative guidance and post-operative confirmation.

[0122] FIG. 27A shows volume-rendered PET/CT demonstrateslocalization of signal in the prostate for pre-operativeplanning.

[0123] FIGS. 27B-27G show white light (left), fluorescence(middle), and composite (right) images obtained at different stagesduring dissection of the prostate.

[0124] FIG. 27B shows detection of fluorescence corresponding toprostate lobes through an intact peritoneum and abdomen.

[0125] FIG. 27C shows fluorescence signal outlines the hK2 positivetissue of the intact ventral prostate lobes.

[0126] FIG. 27D shows a post-hemiectomy: an intact right ventralprostate lobe after left lobe removal.

[0127] FIG. 27E shows imaging after gross removal of both ventrallobes. Bladder indicated with (*).

[0128] FIG. 27F shows delineation of intact dorsal-lateral lobesafter rostral/caudal manipulation of the bladder (*).

[0129] FIG. 27G shows stereoscope magnification (ruler separationsare approximately 800 .mu.m) of area outlined in E. The resectedprostate lobes imaged with (FIG. 27H) conventional white light,(FIG. 27I) fluorescence, and (FIG. 27J) radio-signal.

[0130] FIG. 27K shows a post-surgical PET/CT reveals a smallremnant focus of signal (arrow). After excision at autopsy, seminalvesicles, urethra, and remnant tissue were sectioned and imaged by(FIG. 27L) autoradiography and (FIG. 27M) fluorescentmicroscopy.

[0131] FIG. 27N shows a hematoxylin-eosin stain confirmedadenocarcinoma.

[0132] FIG. 28 shows .sup.18F-FDHT imaging. The distribution at 1.5h after administration in a representative VCaP (arrow) bearingmouse. .sup.18F-FDHT is a radiolabeled analog of thenon-aromatizable dihydrotestosterone. Liver, bile and kidney uptakeis equivalent to or exceeds tumor uptake.

[0133] FIGS. 29A-29B show pathological analysis of sections of GEMmodel of disease after castration.

[0134] FIG. 29A shows 10 .mu.m sections of tissue that did notdemonstrate uptake of 11B6 imaging probe after castration.

[0135] FIG. 29B shows sections from a 11B6 signal-positive tissue.10.times. micrographs (scale is 500 .mu.m) with 40.times. insert(scale is 50 .mu.m). Clockwise from top left: staining for androgenreceptor, Ki-67, haemotoxylin and eosin and c-MYC.

[0136] FIG. 30 shows concordance between quantitative ex vivoimaging and protein content. Uptake of .sup.89Zr-DFO-11B6 on PET(measured as % IA/g in volumes of interest from PET imaging) inindividual prostate lobes correlated to tissue content of hK2(normalized to the total protein concentration). R.sup.2 is0.9928.

[0137] FIG. 31 shows comparison of human and murine 11B6.Humanization of the antibody did not affect the binding and uptakeof radiolabeled antibody in xenograft models of prostate cancer(LNCaP; n=4). Serial microPET images were analyzed for uptake atthe tumor site and in the blood (assessed from manually delineatedvolumes of interest of the xenograft and heart, respectively), andmean volume of interest values are presented as % IA/g.

[0138] FIGS. 32A-32D show h11B6 immunohistochemistry. To evaluate11B6 binding of kallikrein-related peptidase (free hK2),application of the murine 11B6 antibody with an anti-rodentsecondary antibody to human prostate and prostate cancer biopsyspecimens. Hematoxylin counterstained specimens showed theglandular structure of the prostate and hK2 distribution in theprostatic alveoli and intraluminal secretions of representativesamples, including (FIG. 32A) the normal prostate, (FIG. 32B, 32C)two representative prostate tumor tissue specimens, and (FIG. 32D)metastatic foci (lesion in the bone). Scale bar in 4.times. imagesis 250 .mu.m, in 40.times. inserts it is 50 .mu.m.

[0139] FIGS. 33A-33B show schematic representation ofprostate-specific active hK2 in a genetically engineered KLK2expressing mouse model.

[0140] FIG. 33A shows a schematic representation of the generationof the Furin protease activated pre-pro-hK2 GEM to yield aprostate-specific, catalytically active hK2 in vivo. Insertion of aFurin cleavage site sequence upstream of the catalytic region ofpre-pro-hk2.

[0141] FIG. 33B shows that the Furin protease cleavage site isselectively severed by prostate-specific Furin expression,releasing catalytically active hK2.

[0142] FIGS. 34A-34B show genotyping data.

[0143] FIG. 34A shows a southern blot of BAMHI-digested samplesfrom control (lane annotated WT), and transgenic mice hybridizedwith a 2.3 Kb Probasin-fur-hK20-SV40 site probe (annotated 43).This positive founder was used for further breeding. The sizemarkers on the right correspond to BAMHI digested fragments oflambda Hind III, at a dilution corresponding to 10 copies(annotated 10C).

[0144] FIG. 34B shows PCR evaluation of candidate transgenic andcontrol mice for the incorporation of FurhK2 cDNA (upper bands) andGAPDH cDNA (bottom bands) levels indicated equal loading. Lanenumbers refer to individual genotyped animals. Sample number 25correlates to the selected mouse for further breeding (Founder line43). Controls include non-crossed animal (annotated 17), HK2 spiked(31) and FurinhK2 spiked (32) wild type animals. Invitrogen 100base pair ladder shown at right.

DETAILED DESCRIPTION

[0145] It is contemplated that systems, methods, and compositionsof the present disclosure encompass variations and adaptationsdeveloped using information from the embodiments described herein.Adaptation and/or modification of the systems, methods, andcompositions described herein may be performed by those of ordinaryskill in the relevant art.

[0146] Throughout the description, where systems are described ashaving, including, or comprising specific components, or whereprocesses and methods are described as having, including, orcomprising specific steps, it is contemplated that, additionally,there are systems of the present disclosure that consistessentially of, or consist of, the recited components, and thatthere are processes and methods according to the present disclosurethat consist essentially of, or consist of, the recited processingsteps. Moreover, where compositions are described as having,including, or comprising specific components, it is contemplatedthat, additionally, there are compositions of the presentdisclosure that consist essentially of, or consist of, the recitedcomponents.

[0147] It should be understood that the order of steps or order forperforming certain actions is immaterial so long as the processremains operable. Moreover, two or more steps or actions may beconducted simultaneously.

[0148] The mention herein of any publication, for example, in theBackground section, is not an admission that the publication servesas prior art with respect to any of the claims presented herein.The Background section is presented for purposes of clarity and isnot meant as a description of prior art with respect to anyclaim.

[0149] Subject headers are provided herein for convenience only.They are not intended to limit the scope of embodiments describedherein.

[0150] In this application, the use of "or" means "and/or" unlessstated otherwise. As used in this application, the term "comprise"and variations of the term, such as "comprising" and "comprises,"are not intended to exclude other additives, components, integersor steps. As used in this application, the terms "about" and"approximately" are used as equivalents. Any numerals used in thisapplication with or without about/approximately are meant to coverany normal fluctuations appreciated by one of ordinary skill in therelevant art. In certain embodiments, the term "approximately" or"about" refers to a range of values that fall within 25%, 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or less in either direction (greater than or lessthan) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

[0151] "Administration": As used herein, the term "administration"refers to the administration of a composition to a subject orsystem. Administration to an animal subject (e.g., to a human) maybe by any appropriate route. For example, in some embodiments,administration may be bronchial (including by bronchialinstillation), buccal, enteral, interdermal, intra-arterial,intradermal, intragastric, intramedullary, intramuscular,intranasal, intraperitoneal, intrathecal, intravenous,intraventricular, within a specific organ (e.g., Intrahepatic),mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical,tracheal (including by intratracheal instillation), transdermal,vagin*l and vitreal. In some embodiments, administration mayinvolve intermittent dosing. In some embodiments, administrationmay involve continuous dosing (e.g., perfusion) for at least aselected period of time. As is known in the art, antibody therapyis commonly administered parenterally (e.g., by intravenous orsubcutaneous injection).

[0152] "Biomarker": The term "biomarker" is used herein, consistentwith its use in the art, to refer to a to an entity whose presence,level, or form, correlates with a particular biological event orstate of interest, so that it is considered to be a "marker" ofthat event or state. To give but a few examples, in someembodiments, a biomarker may be or comprises a marker for aparticular disease state, or for likelihood that a particulardisease, disorder or condition may develop. In some embodiments, abiomarker may be or comprise a marker for a particular disease ortherapeutic outcome, or likelihood thereof. Thus, in someembodiments, a biomarker is predictive, in some embodiments, abiomarker is prognostic, in some embodiments, a biomarker isdiagnostic, of the relevant biological event or state of interest.A biomarker may be an entity of any chemical class. For example, insome embodiments, a biomarker may be or comprise a nucleic acid, apolypeptide, a lipid, a protein (e.g., an antibody), acarbohydrate, a small molecule, an inorganic agent (e.g., a metalor ion), or a combination thereof. In some embodiments, a biomarkeris a cell surface marker. In some embodiments, a biomarker isintracellular. In some embodiments, a biomarker is found outside ofcells (e.g., is secreted or is otherwise generated or presentoutside of cells, e.g., in a body fluid such as blood, urine,tears, saliva, cerebrospinal fluid, etc.

[0153] "Cancer": The terms "cancer", "malignancy", "neoplasm","tumor", and "carcinoma", are used interchangeably herein to referto cells that exhibit relatively abnormal, uncontrolled, and/orautonomous growth, so that they exhibit an aberrant growthphenotype characterized by a significant loss of control of cellproliferation. Cancer cells include precancerous (e.g., benign),malignant, pre-metastatic, metastatic, and non-metastaticcells.

[0154] "Carrier": As used herein, "carrier" refers to a diluent,adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids,such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water or aqueous solutionsaline solutions and aqueous dextrose and glycerol solutions arepreferably employed as carriers, particularly for injectablesolutions. Suitable pharmaceutical carriers are described in"Remington's Pharmaceutical Sciences" by E. W. Martin.

[0155] "Marker": A marker, as used herein, refers to an entity ormoiety whose presence or level is a characteristic of a particularstate or event. In some embodiments, presence or level of aparticular marker may be characteristic of presence or stage of adisease, disorder, or condition. To give but one example, in someembodiments, the term refers to a gene expression product that ischaracteristic of a particular tumor, tumor subclass, stage oftumor, etc. Alternatively or additionally, in some embodiments, apresence or level of a particular marker correlates with activity(or activity level) of a particular signaling pathway, for examplethat may be characteristic of a particular class of tumors. Thestatistical significance of the presence or absence of a marker mayvary depending upon the particular marker. In some embodiments,detection of a marker is highly specific in that it reflects a highprobability that the tumor is of a particular subclass. Suchspecificity may come at the cost of sensitivity (i.e., a negativeresult may occur even if the tumor is a tumor that would beexpected to express the marker). Conversely, markers with a highdegree of sensitivity may be less specific that those with lowersensitivity. According to the present invention a useful markerneed not distinguish tumors of a particular subclass with 100%accuracy.

[0156] "Peptide" or "Polypeptide": The term "peptide" or"polypeptide" refers to a string of at least two (e.g., at leastthree) amino acids linked together by peptide bonds. In someembodiments, a polypeptide comprises naturally-occurring aminoacids; alternatively or additionally, in some embodiments, apolypeptide comprises one or more non-natural amino acids (i.e.,compounds that do not occur in nature but that can be incorporatedinto a polypeptide chain; see, for example,http://www.cco.caltech.edu/.sup..about.dadgrp/Unnatstruct.gif,which displays structures of non-natural amino acids that have beensuccessfully incorporated into functional ion channels) and/oramino acid analogs as are known in the art may alternatively beemployed). In some embodiments, one or more of the amino acids in aprotein may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a phosphate group, a farnesylgroup, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc.

[0157] "Radiolabel" or "Radionuclide": As used herein, "radiolabel"or "radionuclide" refers to a moiety comprising a radioactiveisotope of at least one element. Exemplary suitable radiolabelsinclude but are not limited to those described herein. In someembodiments, a radiolabel is one used in positron emissiontomography (PET). In some embodiments, a radiolabel is one used insingle-photon emission computed tomography (SPECT). In someembodiments, radioisotopes comprise .sup.99mTc, .sup.111In,.sup.64Cu, .sup.67Ga, .sup.68Ga, .sup.186Re, .sup.188Re,.sup.153Sm, .sup.177Lu, .sup.67Cu, .sup.123I, .sup.124I, .sup.125I,.sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.186Re, .sup.153Sm,.sup.166Ho, .sup.177Lu, .sup.149Pm, .sup.90Y, .sup.213Bi,.sup.103Pd, .sup.109Pd, .sup.159Gd, .sup.140La, .sup.198Au,.sup.199Au, .sup.169Yb, .sup.175Yb, .sup.165Dy, .sup.166Dy,.sup.67Cu, .sup.105Rh, .sup.111Ag, .sup.89Zr, .sup.225Ac,.sup.82Rb, .sup.212Bi, .sup.213Bi, and .sup.192Ir.

[0158] "Sample": As used herein, the term "sample" typically refersto a biological sample obtained or derived from a source ofinterest, as described herein. In some embodiments, a source ofinterest comprises an organism, such as an animal or human. In someembodiments, a biological sample is or comprises biological tissueor fluid. In some embodiments, a biological sample may be orcomprise bone marrow; blood; blood cells; ascites; tissue or fineneedle biopsy samples; cell-containing body fluids; free floatingnucleic acids; sputum; saliva; urine; cerebrospinal fluid,peritoneal fluid; pleural fluid; feces; lymph; gynecologicalfluids; skin swabs; vagin*l swabs; oral swabs; nasal swabs;washings or lavages such as a ductal lavages or broncheoalveolarlavages; aspirates; scrapings; bone marrow specimens; tissue biopsyspecimens; surgical specimens; feces, other body fluids,secretions, and/or excretions; and/or cells therefrom, etc. In someembodiments, a biological sample is or comprises cells obtainedfrom an individual. In some embodiments, a sample is or comprises atumor, tumor tissue, or tumor cells. In some embodiments, obtainedcells are or include cells from an individual from whom the sampleis obtained. In some embodiments, a sample is a "primary sample"obtained directly from a source of interest by any appropriatemeans. For example, in some embodiments, a primary biologicalsample is obtained by methods selected from the group consisting ofbiopsy (e.g., fine needle aspiration or tissue biopsy), surgery,collection of body fluid (e.g., blood, lymph, feces etc.), etc. Insome embodiments, as will be clear from context, the term "sample"refers to a preparation that is obtained by processing (e.g., byremoving one or more components of and/or by adding one or moreagents to) a primary sample. For example, filtering using asemi-permeable membrane. Such a "processed sample" may comprise,for example nucleic acids or proteins extracted from a sample orobtained by subjecting a primary sample to techniques such asamplification or reverse transcription of mRNA, isolation and/orpurification of certain components, etc.

[0159] "Subject": As used herein, the term "subject" includeshumans and mammals (e.g., mice, rats, pigs, cats, dogs, andhorses). In many embodiments, subjects are mammals, particularlyprimates, especially humans. In some embodiments, subjects arelivestock such as cattle, sheep, goats, cows, swine, and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. Insome embodiments (e.g., particularly in research contexts) subjectmammals will be, for example, rodents (e.g., mice, rats, hamsters),rabbits, primates, or swine such as inbred pigs and the like.

[0160] "Therapeutic agent": As used herein, the phrase "therapeuticagent" refers to any agent that has a therapeutic effect and/orelicits a desired biological and/or pharmacological effect, whenadministered to a subject.

[0161] "Treatment": As used herein, the term "treatment" (also"treat" or "treating") refers to any administration of a substancethat partially or completely alleviates, ameliorates, relives,inhibits, delays onset of, reduces severity of, and/or reducesincidence of one or more symptoms, features, and/or causes of aparticular disease, disorder, and/or condition. Such treatment canbe of a subject who does not exhibit signs of the relevant disease,disorder and/or condition and/or of a subject who exhibits onlyearly signs of the disease, disorder, and/or condition.Alternatively or additionally, such treatment can be of a subjectwho exhibits one or more established signs of the relevant disease,disorder and/or condition. In some embodiments, treatment can be ofa subject who has been diagnosed as suffering from the relevantdisease, disorder, and/or condition. In some embodiments, treatmentcan be of a subject known to have one or more susceptibilityfactors that are statistically correlated with increased risk ofdevelopment of the relevant disease, disorder, and/orcondition.

[0162] Targeting the androgen receptor (AR) pathway by receptorblockade and androgen depletion prolongs survival in patients withprostate cancer and a subset of breast cancers, but drug resistancerapidly develops. Understanding this resistance is confounded by alack of non-invasive means to assess AR activity in vivo. Here ispresented an approach involving intracellular accumulation of asecreted antigen targeting antibody (SATA) for diseasecharacterization and therapy. AR-regulated human kallikrein-relatedpeptidase (free-hK2) is a prostate tissue-specific antigen producedin prostate cancer and androgen-stimulated breast cancer cells.Fluorescent and radio-conjugates of 11B6, an antibody targetingfree-hK2, are internalized and non-invasively report AR-pathwayactivity in metastatic and genetically engineered models of cancerdevelopment and treatment. Uptake is mediated by a previouslyunrecognized mechanism involving the neonatal Fc-receptor. Thetechnology described herein transforms the current antibodylandscape by demonstrating cell-specific SATA uptake for diagnosisand therapy in other cancers and/or metastases.

[0163] Presented herein are systems, methods, and compositionsinvolving the use of murine and/or humanized antibodies targetingfree PSA (such as 5A10) and/or free hK2 (such as 11B6) for in vivotargeting of androgen receptor (AR) positive cancer (e.g., breastcancer, e.g., prostate cancer). For example, the antibodies can beused alone (e.g., 5A10 or 11B6) or in combination (e.g., 5A10 and11B6).

[0164] For example, in certain embodiments, the present disclosureis directed to immuno-PET/SPECT and/or immuno-fluoresce-guidedimaging for diagnosing, localizing, radiation dose planning, and/orevaluating therapy response (e.g., anti-androgen receptortherapeutics, surgery and external irradiation) in androgenreceptor (AR) positive breast cancer or PCa. Evaluation can includemonitoring of AR-upregulation of KLK2 and KLK3 in response toexternal irradiation.

[0165] In other embodiments, for example, the present disclosure isdirected to radio-immunotherapy (RIT) treatment of AR-positivebreast cancer by administration (e.g., injection) of a free-PSAand/or free hK2 antibody labelled with a radioisotope after KLK2and KLK3 induction by progesterone, testosterone orirradiation.

[0166] In other embodiments, for example, the present disclosureprovides an antibody-based platform directed to a secreted antigenthat uses Fc-receptor mediated internalization for cancer imagingand therapy.

[0167] In other embodiments, a new approach is described hereinusing an antibody (e.g., 11B6) directed to an epitope accessibleonly on the free, catalytically active form of humankallikrein-related peptidase 2 (hK2). When 11B6 is bound to activehK2, this complex is permanently internalized and transported tolysosomal compartments. Despite the hom*ology similarity between thekallikreins, 11B6 is specific for hK2 and does not bind PSA.Humanized IgG1 11B6 internalizes and accumulates in BCa cells thatexpress human kallikrein 2 (hK2), which is only expressed when theAR-axis is active.

[0168] The antibody 11B6 specifically binds to an epitope in thecatalytic pocket of hK2 that is blocked by protease inhibitors whenthe enzyme is shed/released into the blood circulation. By labelinghumanized 11B6 (hu11B6) with the positron emitting radio-metalZirconium-89 (.sup.89Zr), or other compounds that can be detectedby PET or SPECT, presented herein is an immune-imaging platform(hu11B6) that quantitatively detects presence and activity of theAR axis in BCa. In addition, experiments are also described inwhich 11B6 is labeled with Actinium-225 (.sup.225Ac), an alphaparticle chemical element, for therapeutic applications.

[0169] Uptake of 11B6 is observed in free hK2-producing cancercells, as described in the experiments presented herein, in aprocess facilitated by the neonatal Fc receptor (FcRn). Thisfeature is recapitulated in AR-positive BCa models treated byhormones which stimulate hK2 production. Therefore, the presentdisclosure demonstrates a unique ability to profile and monitor ARactivity in two commonly diagnosed non-cutaneous cancers, PCa andsome types of BCa.

[0170] In certain embodiments, treatment methods are presentedincluding administration of .sup.225Ac-DOTA-hu11B6 in combinationwith induction of the AR-axis by progesterone treatment,testosterone treatment, and/or external irradiation, for bettertherapeutic effect.

[0171] In certain embodiments, 11B6 is applied for both positronemission tomography (PET) and fluorescent imaging in xenograft andgenetically engineered models for disease detection to 1)quantitatively assess AR pathway activity, 2) determinepharmacodynamic parameters, and 3) evaluate treatment efficacy inimmunocompetent models, and 4) guide treatment in clinicallyrelevant scenarios.

[0172] 11B6 immunoimaging resolves issues at key clinical decisionpoints for both prostate and breast cancer patients tosignificantly improve management. Also, it is shown herein that theFcRn mediated uptake mechanism can be exploited to facilitateuptake by other SATA. There does not appear to be any previousreport of targeted tissue specific uptake of a secreted antigen;thus, the technology described herein provides a new strategy forprecision imaging of disease processes.

Results: Uptake of hK2-Targeting .sup.89Zr-11B6 RadiotracerCorrelates to Expression Level of its Target Enzyme

[0173] Studies have revealed that hK2 is an anatomically anddisease restricted protein. Described herein is a generated murineantibody, 11B6, with specificity for the catalytic pocket of freehK2. Conjugated to desferrioxamine B (DFO) and subsequentZirconium-89 (.sup.89Zr) labeling yielded .sup.89Zr-11B6, apositron emission tomography (PET) radiotracer. A competitionbinding assay was conducted revealing that bioconjugation of 11B6resulted in no significant loss of affinity for hK2 (FIG. 10). Invitro studies of .sup.89Zr-11B6 uptake showed expression-specificuptake, and specificity was verified by blocking with excess 11B6.Activity after washing revealed this SATA was internalized by hK2expressing cells.

[0174] To test if uptake occurred in vivo, the dose of.sup.89Zr-11B6 was first optimized with an escalation study and PETquantification in mice bearing human prostate cancer xenografts(FIGS. 1A-1D). The time to tumor saturation inversely correlatedwith tracer mass and improved tumor/blood contrast; 2.4, 4.2, 7.7and 13.7 hours for 300, 150, 50, and 15 .mu.g of .sup.89Zr-11B6,respectively. Subsequent experiments utilized 50 .mu.g.sup.89Zr-11B6, which achieved tumor saturation with low backgroundactivity after 120 hours (FIG. 1C). In vivo specificity wasverified by blocking (1 mg, unlabeled-11B6) and using hK2-negativeDU145 xenografts. To assess the potential for imaging of patientsthat have failed hormone therapy, uptake was measured in castrationresistant 22Rv1 tumor xenografts, as well. Here, it was observedthat there was robust localization to the tumor through continuedAR-driven hK2 expression (FIG. 11).

[0175] KLK2 expression was evaluated in 7 xenograft lines (FIG.12A). VCaP exhibited the highest KLK2 levels and showed markedlyhigher .sup.89Zr-11B6 internalization (80.7 percent injectedactivity per gram (% IA/g)) compared to LNCaP (24.7% IA/g) (FIG.1D), demonstrating the ability to determine hK2 expression statusin vivo. The expression level of KLK2 did not correlate with twoother AR-governed imaging targets, KLK3 (PSA) or FOLH1 (PSMA),underlining the use of hK2 as a distinct biomarker (FIGS.12A-12D).

Delineating Bone Metastases

[0176] Bone is a common site of PCa and BCa metastasis, oftenmanifesting a mixed bone forming/resorbing phenotype thatcomplicates detection by current clinical imaging methods, whichrely on the uptake at sites with increased osteoblastic activity.The ability of .sup.89Zr-11B6 to detect both phenotypes wasevaluated using intraosseous LNCaP-AR (osteolytic) and VCaP(osteoblastic) bone metastases models with control PC3 (AR/hK2negative osteolytic) bone lesions (FIGS. 2A-2C). .sup.89Zr-11B6 PETdemonstrated robust delineation in both osteometastatic phenotypesof AR-positive disease, with uptake delayed relative tosubcutaneously inoculated tumors (FIGS. 13A-13B).

[0177] Faithful recapitulation of PCa for study in mice isparticularly difficult given the absence of murine orthologs ofseveral human prostate specific genes, including the prostatekallikreins. To test tracer kinetics in an immunocompetent milieuand measure uptake in autochthonous mouse tumors, a prostatefull-length KLK2 construct encoding pre-pro-hK2 was cloned undercontrol of the probasin promoter (Pb_KLK2), enablingprostate-specific and androgen driven expression of hK2. Using B6mice as negative controls, .sup.89Zr-11B6 uptake was specific tohK2 positive prostatic tissue in vivo (FIGS. 14A-14C).

11B6-hK2 is Internalized by FcRn

[0178] To investigate SATA internalization, conjugated 11B6 wasfirst evaluated in whole-mount sections of prostate tissue fromPb_KLK2 mice. A high concordance between intravenously administeredfluorescent and radioactive tracer was observed, as was anassociation between antibody uptake and staining for AR (FIGS.3A-3E). 11B6 in the lumen of prostatic ducts suggested uptake byepithelial cells, confirmed by confocal microscopy (FIG. 3B). Toverify, single cells extracted from this tissue were analyzed forfluorescent antibody uptake. In addition, analysis of PCa celllines was performed in vitro (FIGS. 15A-15D).

[0179] The neonatal Fc receptor (FcRn) generally facilitatesantigen recognition in luminal structures throughout the body andis expressed in a large set of PCa lines (FIG. 12D). Intracellulartransport of the conjugate was determined by co-staining prostatecancer cells for FcRn and anti-IgG. Following pulsed exposure, 11B6is associated with FcRn during the early phase of uptake. At latetime-points, 11B6 appears intracellularly, and FcRn returns to thecell membrane. The 11B6-hK2 complex is shuttled from physiologicalpH early-endosomes to acidic late-endosomes, as shown using apH-responsive dye conjugated to 11B6 and imaged in live cells(FIGS. 3F, 3G).

[0180] To confirm the specific role of FcRn in internalization ofthe SATA-antigen complex, recombinant mutant-11B6 IgG.sub.1 wasgenerated (modified at Histidine 435 to Alanine; H435A-11B6) toabrogate FcRn binding and cellular uptake was compared inphysiological and acidified media. This mutation abrogates FcRnbinding but does not affect variable region recognition oraffinity. Surface plasmon resonance affinity of FcRn for 11B6 waspH dependent, and absent in the H435A-11B6 mutant (FIG. 16A). Inculture, incubation at acidic pH conditions, as found in tumors andthe prostate, augmented internalization (FIGS. 16A-16B). Thepresence of both FcRn and hK2 was required for internalization, andisotype matched control antibody did not bind cells or transportintracellularly via endosomes (FIG. 18).

[0181] .sup.89Zr-labeled 11B6 and mutant-Fc antibody were appliedto establish FcRn dependence in vivo. Uptake of the mutant-Fcantibody matched that of control non-specific IgG (shown for LNCaP;(FIGS. 17A-17B), despite retained immunoreactivity of the FcRnbinding-deficient antibody (FIG. 17C). Relative to the wild-type11B6 antibody, H435A-11B6 uptake in immunodeficient xenograftmodels was significantly lower (21.2% IA/g for VCaP, P=5.97E-5; and5.23% IA/g for LNCaP, P=8.24E-7); (FIG. 17D). Immunocompetent GEMMof adenocarcinoma (obtained by crossing Pb_KLK2 withARR2/probasin-Myc (Hi-Myc), accumulate 11B6 in the transformed lobeof the prostate, while uptake of FcRn-binding deficient H435A-11B6was abolished (FIGS. 17A-17E).

[0182] FcRn is widely expressed in tissues throughout the body, andparticularly concentrated in the liver. Antibody imaging in thisorgan is difficult as non-specific uptake and clearance increasebackground. Thus, in addition to the demonstration of changes inuptake in GEM presented herein (FIGS. 17A-17E), it was desired totest if metastasis of PCa to the liver could be identified, anend-stage site of disseminated disease. .sup.89Zr-11B6 PET andmagnetic resonance imaging revealed specific focal accumulation inhK2-expressing LREX' metastases in the liver that were resistant toenzalutamide, a second-generation AR antagonist (FIG. 18).Autoradiography and histopathological findings correlate with thenoninvasive assessment, demonstrating that targeting a secretedtarget downstream of central PCa biology is able to quantitateincipient resistance.

[0183] It was also tested whether uptake of antibody-secretedantigen complexes could be applied to other targets. Previously, itwas shown that targeting free-PSA with an antibody (5A10) candelineate subcutaneous xenografts (for example, U.S. Pat. No.8,663,600 describes a method involving injection of tracer-labelledantibodies, and visualizing PSA-producing or hK2-producing tissuefor diagnosis of prostate cancer). Transient uptake was observed,as the SATA-antigen complex was not internalized and washed out ofthe tumor microenvironment. Previously identified residues at theconstant heavy chain 2 and 3 (CH2/CH3) junction contribute to thepH-dependent affinity of the IgG interaction with FcRn, yieldingmultiple options for the inability of 5A10 to bind FcRn. Thecomplementarity determining regions (CDRs) of 5A10 were graftedonto the 11B6 Fc-scaffold (bearing the histidine at residue 435;5A10.sup.H435-wt) In vivo, steadily increasing tumor uptake wasobserved using .sup.89Zr-- 5A10.sup.H435-wt in LNCaP xenografts, incontrast to the original PSA-targeting 5A10 (FIGS. 19A-19G).

Antibody-hK2 Uptake in AR.sup.+ Breast Cancer

[0184] KLK2 expression is restricted to the prostate and PCatissues in man, however it has been demonstrated that hK2 and PSAare detectable in (female) BCa cell lines and primary patientsamples after appropriate activation of the AR-pathway by steroidhormones. Experiments were performed to investigate whetherFcRn-mediated internalization of the antibody-bound hK2 is prostatespecific. Under dihydrotestosterone (DHT), a subset of AR-positiveBCa lines secrete hK2, including the triple-negative BCa lineMFM-223 (FIG. 20). Androgen stimulation increased the AR-responsiveKLK2 (FIGS. 21C-21D).

[0185] It was assessed whether 11B6 is internalized in anon-prostate derived cancer model. .sup.89Zr-11B6 was used to imageAR-positive BCa with BT474 (ER.sup.+/PR.sup.+/HER2.sup.+/AR.sup.+)xenografts. .sup.89Zr-11B6 uptake was significantly greater in DHTtreated female mice, compared to estrogen alone (P=0.001, FIG.19E). As above, confocal microscopy reveals BT474 cells with andwithout DHT treatment internalize 11B6 in a time dependent manner(FIGS. 21A-21E). FIG. 22 is a PET/CT image of .sup.89Zr-DFO-11B6 ina subcutaneous MFM223 model following DHT stimulation, revealingthe presence of AR+ triple negative breast cancer.

Staging Adenocarcinoma and Monitoring Treatment

[0186] Next, .sup.89Zr-11B6 PET was applied to detect and monitortumor progression in the prostate of transgenic models ofadenocarcinoma (FIG. 4). Greater SATA uptake at sites of disease isnoted, demonstrating heterogeneous progression, even at the smallscale of the mouse prostate. Quantitation of tracer accumulation inthe prostate corresponded with transformation from prostaticintraepithelial neoplasia through to adenocarcinoma. Ex vivoautoradiography of tracer microdistribution and histologicaladenocarcinoma is shown for a 50-week-old mouse (FIG. 23).

[0187] Use of the anti-hK2 tracer to assess AR-activity in responseto intervention was studied in three clinical scenarios thatcurrently lack (but would greatly benefit from) molecularlyspecific assessment. In the first sub-study, .sup.89Zr-11B6 wasmeasured before and after surgical castration in a bone metastasismodel using LNCaP-AR/luc (expressing luciferase under the controlof ARR2-Pb). Standard-of-care blood measurements of PSA and.sup.18F sodium fluoride (18 imaging. .sup.89F--NaF) PET bone scanswere compared to hK2-targeted PET Zr-11B6 uptake decreasedfollowing castration (P=0.005; FIGS. 5A-5C), as did AR-drivenluciferase (P=0.0012; FIG. 5D). Conventional metrics of prostatecancer bone lesion response, PSA and .sup.18F--NaF, remainedunchanged (FIGS. 5E-5F).

[0188] The second scenario simulated intermittent androgendeprivation (IAD) therapy. There is debate concerning the optimaltreatment regime (between intermittent or continuous inhibition)for hormonal therapy. Pb_KLK2 XHi-Myc mice received depotinjections of Degarelix, a gonadotrophin-releasing hormone (GnRH)antagonist, ablating androgen production for 2 months..sup.89Zr-11B6 imaging was performed longitudinally to assessresponse to androgen deprivation, as well as reactivation followingdiscontinuation. .sup.89Zr-11B6 decreased following castration butreemerged at the end of the treatment period, enabling readout ofpharmacodynamic inhibition of the AR pathway (FIGS. 24A-24E).

[0189] A final clinical simulation involved non-invasive imaging ofthe impact of different degrees of inhibition on AR activity in thetumor (intratumoral) and prostate itself (intraprostatic).Progression of disease was initially monitored in 14 Pb_KLK2XHi-Myc mice using .sup.89Zr-11B6 PET. Thereafter, mice wererandomized into 3 treatment groups: vehicle (n=4), castration(n=6), or castration plus Enzalutamide (n=4). Substantialheterogeneity was noted in individual subject's (e.g., animal's)prostatic uptake of the tracer during progression and in responseto therapy (FIGS. 6A-6C).

[0190] SATA uptake was repressed during the last months oftreatment in mice receiving adjuvant AR blockade, indicating abenefit for adjuvant AR blockade using anti-androgens in thepost-castration setting (FIGS. 6D, 6E). Reverse transcriptionpolymerase chain reaction (RT-PCR) analysis of prostatic tissueharvested from the mice receiving AR-blockade displayedsignificantly lower KLK2 expression compared to other groups(P=0.0016 for castrate alone, and P=0.0089 for castration andenzalutamide; FIG. 26A). Expression differences with and withoutadjuvant therapy were small, as were between the lobes of theprostate containing focal sites of uptake from those that werenegative (FIG. 26B). KLK2 This islikely due to the fact that RT-PCRreflects an average of the expression based on the whole lobe(FIGS. 25A-25E). However, the hK2 concentration in prostatic tissuelysate indicated a strong positive correlation between.sup.89Zr-11B6 uptake and AR-dependent hK2 production (FIG. 29),and immunopathology for proliferation marker Ki67 and AR (FIG. 30)reveal sub-regions that continue to proliferate following treatmentwhich are selected by focal 11B6-signal. (FIGS. 23, 26A-26C).

Directing Treatment in Real-Time

[0191] Radio- and fluorescently-labeled tracers indicated highlyspecific uptake in the cells of the prostate for noninvasiveassessment (FIG. 3). To demonstrate the value of 11B6 imagingprostatic expression in the translational setting, the fulltreatment course was simulated to encompass pre-, intra-, andpost-operative clinical decision points using dual-labeled.sup.89Zr-DFO and Cy5.5 for PET and fluorescence. This concept wasexplored using the Pb_KLK2.times.Hi-Myc model.

[0192] PET was performed to assess disease burden (FIG. 27A), whichwas then resected using a fluorescent surgical stereoscope forreal-time guidance (FIGS. 27A-27G). Remnant prostatic tissue washarvested to confirm margins, and excised tissues were scanned forfluorescent and radio signals and hK2 protein (FIGS. 27H-27J).After removing fluorescent tissues, peritoneum and skin weresutured, and a post-operative PET was acquired (FIG. 27K). A regionof tracer accumulation could be identified by post-operative PET/CTand was subsequently removed at autopsy. This was confirmed to beprostate tissue with fluorescence microscopy, autoradiography, andhistochemistry (FIGS. 27L-27N).

Humanized 11B6 and Toxicity

[0193] For intended use in humans, the rodent CDRs were graftedinto a human immunoglobulin framework to yield hu11B6, withoutadverse effects on binding affinity or specificity. Surface plasmonresonance-determined dissociation and association rate constantsfor all versions of 11B6 were calculated to be in the range of 10-5(koff) and 105 M-1 s-1 (kon), respectively. No statisticaldifference in the apparent affinity was observed between hu11B6 andits DFO conjugate (FIG. 10).

[0194] The kinetics and accumulation of the humanized conjugate,.sup.89Zr-DFO-hu11B6, were not significantly different from themouse IgG1 version of 11B6 (FIG. 31). The affinity and favorabletoxicity of this internalized SATA give it considerabletranslational potential. Finally, to assess the capacity to bindhK2 in human tissues, 11B6 was applied to human tissue specimens.The hK2 distribution in normal prostate, prostate adenocarcinoma,and a bone lesion can be identified by 11B6 immunodetection (FIGS.32A-32D).

Discussion

[0195] The ability to detect malignant cells, to monitorpathological processes, or to deliver therapeutic compounds isneeded to improve PCa and TN-BCa management. Extracellularcytokines and proteins are recognized as important mediators ofthese diseases, and have been widely targeted with antibodies tocombat disease or ameliorate its symptoms. However, biologicsdirected to these extracellular components have not enabledcellular targeting for imaging or treatment, limiting the abilityto affect diseased cells themselves. Here, it is reported that ananti-hK2 antibody, 11B6, enables cell-specific accumulation ofdiagnostic and therapeutic agents to the most common invasivecancers in men and women.

[0196] Uptake of 11B6 in hK2-expressing tissues was FcRn-mediated,which is a unique demonstration of antibody-antigen internalizationby cells which themselves express the target. FcRn enables passivetransfer of IgG from mother to offspring in the early stages oflife as well as a variety of physiologic functions in adultimmunity. Notably, FcRn facilitates transport of IgG.sub.1 andrecycling of IgG-immune complexes across otherwise impermeablepolarized epithelia. 11B6 exploits this mechanism, resulting incellular accumulation of an immune complex which avoids theprecipitous washout observed using a previous kallikrein-targetedconstruct. The wider applicability of this approach to enable cellspecific accumulation of a second SATA to PSA (5A10.sup.H435-wt) isdemonstrated herein. FcRn binding is pH dependent and the lower pHat sites of disease may provide an even more favorablemicroenvironment to generate imaging contrast compared tonon-malignant tissue. These results have immediate relevance forboth PCa and BCa directed imaging and therapy and more widely as astrategy to improve both the magnitude and localization ofinternalizing SATA.

[0197] hK2 has traditionally been evaluated as a prostatebiomarker; however, shown herein is uptake of .sup.89Zr-11B6 inAR-positive breast cancer xenografts under hormone stimulation.Questions surround the repercussions of AR status in BCa. Whileseveral studies implicate a role for AR in pathways that negativelyimpact survival, a correlation between AR and positive prognosticmarkers has also been identified. The application of androgenantagonists in AR-positive BCa indicates that AR inhibition may bebest directed towards basal (triple-negative) rather than luminal Btype/HER2 refractory subtypes. Without wishing to be bound to anytheory, trials suggest that this may represent a new approach totreat TN-BCa. The 11B6 platform enables further study of thenuanced role of AR in the biology of breast cancer by offering theability to guide and monitor treatment.

[0198] The multimodal methods employed against a range of modelsdemonstrate an approach which eliminates long-standing impedimentsto non-invasively monitor disease biology and assist development ofnovel androgen receptor-targeted therapies as a pharmacodynamictool. Biopsy is used in PCa and BCa disease assessment to providedirect readout of tissue organization but is restricted in time,access and accuracy. Conventional imaging to guide biopsy(ultrasound, computed tomography (CT) and MRI) suffers from modestsensitivities for detection and staging, with complication risks.If lesions are detectable, a direct biopsy can provide informationon cellular processes, but is invasive, costly and difficult torepeat.

[0199] In contrast, .sup.89Zr-11B6 PET provides whole-body imagingof disease foci and provides a readout of AR activity for bothprimary and metastatic lesions. In a transgenic c-Myc driven modelof adenocarcinoma, AR-activity was longitudinally evaluated duringdisease progression from the pre-malignant prostate through highdisease burden (FIG. 4). The dynamics of androgen inhibition, forexample with metronomic chemical castration, can be monitoredquantitatively. This imaging platform can be extended to evaluatetreatment regimens, which revealed low levels of AR-pathwayreactivation at sub-organ resolution and enabled a comparisonbetween models of surgical castration versus castration plusadjuvant therapy (FIGS. 6A-6E). The agent may also be used to guidetreatment in real-time or assist in treatment delivery.

[0200] .sup.89Zr-11B6 targets tumorous lesions themselves, ratherthan sites of remodeling, and is able to identify both osteoblasticand osteoclastic metastases (FIGS. 2A-2C). Conventional.sup.18F--NaF bone scans have high sensitivity but lack specificityfor disease, confounding the readout of disease burden especiallypost-therapy. The enhanced precision of treatment monitoring bySATA will help to accelerate preclinical and translational researchtowards answering critical clinical questions for optimal patientcare.

[0201] The technology presented here has direct application in PCaand BCa patients. Humanized-11B6 retains binding characteristics ofthe original agent (FIGS. 25A-25E). The technology is applicable toindividualized patient stratification and management at themolecular level. The approach of designing SATA which facilitatecellular uptake may be relevant to the detection, monitoring, andtreatment of a wide variety of diseases and conditions.

[0202] FIGS. 7A-7B present graphs that show an AR increase afterirradiation of two AR-positive BCa cell lines (BT474 and MFM223).The change in KLK2 and KLK3 is shown for both BT474 and MFMfollowing irradiation.

[0203] FIG. 8 shows a survival graph after injecting.sup.225Ac-DOTA-hu11B6 in DHT-stimulated (i.e. Expression of KLK2)and in non-DHT stimulated mice (i.e. Non-KLK2 expression). FIGS.7A-7B and FIG. 8 demonstrate the value of KLK2 and KLK3induction--e.g., by administration of progesterone, testosterone,and/or, as shown here, by irradiation--prior to administration of afree-PSA and/or free hK2 antibody labelled with a radioisotope forradio-immunotherapy (RIT) of AR-positive breast cancer, inaccordance with an illustrative embodiment of the invention.

[0204] Table 1 shows dissociation rate constants (k.sub.off) form11B6, hu11B6, and DFO-conjugated hu11B6. Based on the twomeasurement series taken for each antibody, no significantdifference in the dissociation rate constants (k.sub.off) was foundbetween the hK2 targeting antibodies.

TABLE-US-00001 TABLE 1 Antibody k.sub.off (10.sup.-5s.sup.-1)Fc2k.sub.off (10.sup.-5s.sup.-1)Fc3 k.sub.off (10.sup.-5s.sup.-1)Fc4Mean Std dev m11B6 1.9 4.9 -- 3.4 .+-.2.1 hu11B6 6.4 6.9 -- 6.7.+-.0.4 hu11B6-DFO 5.8 5.5 -- 5.7 .+-.0.2

[0205] Table 2 shows average association rate constant based on15-18 measurements for each version of 11B6. Differences in rateconstants (k.sub.on) of the tested antibodies were notsignificant.

TABLE-US-00002 TABLE 2 No. Of expts Mean k.sub.on Antibody fitted(10.sup.5M.sup.-1s.sup.-1) Std dev m11B6 18/18 2.48 .+-.0.85 hu11B615/18 1.17 .+-.0.38 hu11B6-DFO 18/18 1.11 .+-.0.22

[0206] Table 3 shows dissociation rate constants (K.sub.D) for thetested antibodies.

TABLE-US-00003 TABLE 3 Antibody Mean K.sub.D 10.sup.-11 M Std devm11B6 19 .+-.15 hu11B6 65 .+-.25 hu11B6-DFO 54 .+-.13

[0207] Table 4 shows .sup.89Zr-11B6 biodistribution and the effectof blocking with cold antibody. Biodistribution values for eachorgan are shown as percent injected activity per gram at 320 h fordifferent cell lines. Data are shown as mean.+-.standard deviationwith n.gtoreq.3.

TABLE-US-00004 TABLE 4 Blocked LNCaP VCaP DU145 (LNCaP) Organ Avg..+-. Avg. .+-. Avg. .+-. Avg. .+-. Blood 3.05 0.61 2.28 1.11 6.481.90 4.01 1.25 Tumor 24.72 2.41 80.68 15.34 1.25 0.58 6.42 2.91Heart 1.60 0.19 0.98 0.20 2.47 0.26 2.46 0.63 Lung 3.60 0.50 2.080.37 5.16 0.66 3.81 1.74 Liver 13.80 2.47 11.64 3.97 16.77 1.0112.82 0.37 Spleen 6.56 2.86 8.58 2.00 4.39 0.33 6.33 1.08 Stomach0.29 0.10 0.31 0.22 0.86 0.35 0.27 0.08 Sm. Intest. 0.52 0.07 0.480.14 3.37 4.45 0.41 0.07 Lg. Intest. 0.42 0.05 0.51 0.17 0.81 0.380.36 0.10 Kidneys 4.10 0.08 1.96 0.37 5.00 0.35 4.59 1.23 Muscle0.38 0.16 0.44 0.12 0.60 0.13 0.57 0.24 Bone 5.52 2.18 1.40 0.561.23 0.32 6.33 3.42

[0208] Table 5 shows receptor status of breast cancer cell linesand secretion of hK2 in response to DHT. The status of estrogen andprogesterone receptor and HER2 amplification, as well as thepresence of AR for common breast cancer cell lines are given. These13 BCa cell lines were tested by immunofluorimetric assay for thepresence of hK2 protein secretion in culture supernatant. No cellsproduced the kallikrein without hormone stimulation, and onlyAR-positive cell lines were found to produce hK2 after the additionof the hormone.

TABLE-US-00005 TABLE 5 Hormone status hK2 Human Andro- producedbreast cancer Estrogen Progesterone gen after AR/PR line receptorreceptor HER2 receptor stimulation AU565 - - + - - BT-20 - - - - -BT-474 + + + + + HCC1806 - - - - - MCF7 + + - - - MDAMB361 + + + -- MDAMB415 + - - - - MDAMB435 - - - - - MDAMB468 - - - - - MFM-223- - - + + SK-BR-3 - - + - - T-47D + + - + + ZR-75-30 + - + - -

[0209] Table 6 shows data values from PET, bioluminescence andclinical chemistry measurements. The data is shown for each grouppre- and post-castration, appended with average and standarddeviation computations. Insufficient bloods for two animals in thePSA assay reduce the group size for this measurement to n=4.

TABLE-US-00006 TABLE 6 Measurement (units) Average .sup.89Zr-11B6Radiance Total PSA 18F-NaF (mean % IA/g) (p/s/cm2/sr) (ng/mL) (mean% IA/g) Post- Post- Post- Post- Untreated Castration UntreatedCastration Untreated Castration Untreated Castration 14.647826.861449 10640000 86260 1.1595 7.26063 7.077255 25.77738 8.70757711030000 766700 4.0825 6.85531 7.534436 19.49468 9.22105 5439000289600 3.841 2.5045 8.851907 10.70157 13.95731 6.919245 900480084100 2.405 3.4995 9.11639 8.213374 24.19821 7.811357 12136000345910 10.539 18.9705 7.650328 7.01773 Average: 18.4693 7.92739028450 306665 5.216875 6.5335 7.946913 8.108873 SD: 5.3777 1.05422608328 278957 3.62452269 8.346592958 0.992123319 1.526253977

Materials and Methods

[0210] Study Design

[0211] The present disclosure investigates the capacity of anantibody targeting the catalytically active site of aprostate-specific protease (in man) to delineate and guidetreatment of primary and metastatic prostate and breast cancer.Binding properties and cellular interaction were evaluated in vitroand in vivo using fluorescent and radio conjugates. Theinternalization of this antibody, via the neonatal Fc receptor,following interaction with its secreted targeted antigen, wasstudied in detail and evaluated in a second antibody targetinganother secreted antigen. Appending the positron-emittingzirconium-89 to the antibody for immunoPET was studied insubcutaneous, osseous and hepatic metastatic, and geneticallyengineered autochthonous prostate cancer models. Tumor uptake anduptake kinetics were measured using manually defined regions ofinterest at multiple time points from 4 h through to 320 h. Imagingstudies in bone and GEM systems were designed to measure treatmenteffect on AR-activity with surgical and/or chemical castration.Breast cancer cell lines were evaluated for KLK2 expression and hK2production with and without hormone stimulation. To study BCa hk2production in vivo, BT474 xenografts with and without androgenstimulation were imaged by .sup.89Zr-11B6. Quantitative in vivo PETimaging data was assessed in addition to ex vivo autoradiographyand gamma counting. PET study duration was sufficiently long toachieve 20E6 coincident events, Cohorts in treatment groups wererandomized and no outliers were excluded.

[0212] All chemicals and reagents of the highest available puritywere purchased from ThermoFisher Scientific, unless otherwisenoted. Murine 11B6 was provided by Dr. Kim Pettersson, Universityof Turku, Finland, while humanized 11B6 (hu11B6) was developed byDiaProst Inc., Lund, Sweden and produced by Innovagen Inc., Lund,Sweden. Enzalutamide (MDV3100), manufactured by Medivation, wasprovided Dr. Charles Sawyers at MSKCC.

[0213] Preparation of Zirconium-89

[0214] Zirconium-89 was produced through the .sup.89Y(p,n).sup.89Zrtransmutation reaction on an EBCO TR19/9 variable-beam energycyclotron (Ebco Industries, Inc.) in accordance with previouslyreported methods. .sup.89Zr-oxalate was isolated in highradionuclidic and radio-chemical purity >99.9 with an effectivespecific activity of 195 to 497 MBq/.mu.g (5.27-13.31 mCi/.mu.g).Immediately prior to radiolabeling, .sup.89Zr[Zr]oxalate wasneutralized with aliquots of NaCO.sub.3 (1 M) to pH 7.

[0215] Preparation of Radiolabeled Construct

[0216] Prior to conjugation, all antibodies were exchanged into2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES; 0.1M, pH 8) by repeated ultracentrifugation (Amicon Centriplus YM-50,Millipore) and gel purification (PD10, GE Healthcare). Thezirconium chelator, Deferoxamine-p-SCN (Areva Med) was conjugatedto the antibody using a molar excess of 7:1. After addition of thebifunctional chelate the reaction was pH adjusted to pH 8.5 withNa.sub.2CO.sub.3 shaken at 37.degree. C. for 1 hour, then purifiedby repeated centrifugation as above, into phosphate buffered saline(PBS). DFO-conjugated 11B6 (400 .mu.L) was mixed with neutralized.sup.89Zr[Zr] and mixed gently. The pH after mixture wascross-checked and adjusted to pH 7, if needed. The labelingreaction was allowed to proceed for 1 hour. The conjugate was thenpurified by repeated purification by ultrafiltration into sterilesaline. Radiochemical yield was assessed after purification averageyield was between 40% and 50%. Radiopurity was assessed byradio-instant thin layer chromatography. Briefly,.sup.89Zr-DFO-11B6 (.sup.89Zr-11B6) was blotted (1 .mu.L) onsilica-impregnated paper and eluted with a solution of 50 mMdiethylenetriaminepentaacetic acid. All labeling reactions achieved>99% radiochemical purity. Average specific activity of thefinal radiolabeled conjugate was 1.4 mCi/mg.

[0217] Preparation of Fluorescently Labeled Constructs

[0218] Prior to conjugation, all antibodies were purified as above.The near infrared fluorophore Cy5.5-NHS (GE Healthcare) wasresuspended in methanol aliquoted and dried using a speedvac. Usinga molar excess of 3:1, the antibody was labeled and the pH wasadjusted to 8.5 with Na.sub.2CO.sub.3. The reaction was shaken at22.degree. C. for 4 hours, followed by gel purification (PD10) andultrafiltration (Amicon). The number of dye molecules per antibodywas evaluated using a spectrophotometer and calculated to be 1.3(SpectraMax M5, Molecular Devices). The dye labeled antibodyconjugate was prepared fresh for each experiment.

[0219] Cell Lines

[0220] LNCaP, DU-145, CWR22Rv1, MDAPCa2b, VCaP were purchased fromAmerican Type Culture Collection. The cell lines were culturedaccording to the manufacturer's instructions. LAPC4, LREX' andLNCaP-AR-luc was previously developed and reported by the Sawyerslaboratory.

[0221] Animal Studies

[0222] All animal experiments were conducted in compliance withinstitutional guidelines at Memorial Sloan-Kettering Cancer Center.For xenograft studies: male athymic BALB/c (nu/nu) mice (6-8 weeksold, 20-25 g) were obtained from Charles River. LNCaP, DU-145,CWR22Rv1, MDAPCa2b, LAPC4, and VCaP tumors were inoculated in theright flank by subcutaneous injection of 1-5.times.10.sup.6 cellsin a 200 .mu.L cell suspension of a 1:1 v/v mixture of media withMatrigel (Collaborative Biomedical Products, Inc.). Tumorsdeveloped after 3 to 7 weeks. Enzalutamide (ENZ, MDV3100) wasdissolved in dimethyl sulfoxide (DMSO) so that the final DMSOconcentration when administered to animals would be 5. Theformulation of the vehicle is 1 carboxymethyl cellulose, 0.1polysorbate 80, and 5 DMSO. Enzalutamide or vehicle wasadministered daily by gavage. Liver xenografts of the LREX' linewere implanted.

[0223] Flank xenografts of the BT474 cell line were establishedusing established procedures. Briefly, 17.beta.-estradiol pellets(0.72 mg/pellet) (Innovative Research of America, Sarasota, Fla.)were inserted subcutaneously prior to inoculation of1.times.10.sup.6 cells in a 200 .mu.L suspension of a 1:1 v/vmixture of media with Matrigel (n=6). For this study, female Balb/cnu/nu animals were used. Animals in the DHT-positive group weresupplemented with an additional subcutaneous 12.5 mg DHT pellet(Innovative Research of America).

[0224] Preparation of Osseous Tumor Grafts

[0225] Male CB-17 severe combined immunodeficient (SCID) mice (6-8weeks old) were anesthetized with a mixture of ketamine/xylazine,and a parapatellar incision was made in the left hindlimb. Thetibia was punctured using a needle, and 1.times.10.sup.5 cells(VCaP-luc or LNCaP-AR) were injected into the cavity. The puncturewas closed with bone wax, the incision sutured, and animalsreceived a palliative dose of carprofen (5 mg/kg) once daily for 3days post inoculation. Tumor development was followed withbioluminescence imaging and confirmed with CT.

[0226] Biodistribution Studies

[0227] Biodistribution studies were conducted to evaluate theuptake of .sup.89Zr-11B6 in human prostate cancer xenograft models.Mice received .sup.89Zr-11B6 [3.7-5.55 MBq (100-150 .mu.Ci), 300,100, 50, or 15 .mu.g of protein, in 150 .mu.L sterile saline forinjection] through intravenous tail-vein injection (t=0 hour).Animals (n=4-5 per group) were euthanized by CO.sub.2 asphyxiationat 24, 72, 96, 120, 240 and 344 hours post-injection and blood wasimmediately harvested by cardiac puncture. Eleven tissues(including the tumor) were removed, rinsed in water, dried onpaper, weighed, and counted on a gamma-counter for accumulation of.sup.89Zr radioactivity. Count data were corrected for backgroundactivity and decay and the tissue uptake [measured in units ofpercentage injected activity per gram (% IA/g)] for each sample wascalculated by normalization to the total amount of activityinjected.

[0228] Small-Animal Positron Emission Tomography Imaging

[0229] PET imaging experiments were conducted on a micro-PET Focus120 scanner (Concorde Microsystems). In initial studies, mice (n=4)were administered formulations of .sup.89Zr-11B6 [3.7-5.55 MBq(100-150 .mu.Ci), 300, 100, 50, or 25 .mu.g of protein, in 150.mu.L sterile saline for injection] through i.v. Tail-veininjection. Approximately 5 minutes before recording PET images,mice were anesthetized by inhalation of 1% to 2% isoflurane (BaxterHealthcare)/oxygen gas mixture and placed on the scanner bed. PETimages were recorded at various time points between 1 and 344 hourspost-injection. List-mode data were acquired using a .gamma.-rayenergy window of 350 to 750 keV and a coincidence timing window of6 nanoseconds. PET image data were corrected for detectornon-uniformity, dead time, random coincidences and physical decay.For all static images, scan time was adjusted to ensure between15-25 million coincident events were recorded.

[0230] Data were sorted into 3-dimensional histograms by Fourierrebinning, and transverse images were reconstructed using a maximuma priori algorithm to a 256.times.256.times.95(0.72.times.0.72.times.1.3 mm) matrix. The reconstructed spatialresolution for .sup.89Zr was 1.9 mm full-width half-maximum at thecenter of the field of view. The image data were normalized tocorrect for non-uniformity of response of the PET, dead-time countlosses, positron branching ratio, and physical decay to the time ofinjection, but no attenuation, scatter, or partial-volume averagingcorrection was applied. An empirically determined systemcalibration factor [in units of (mCi/mL)/(cps/voxel)] for mice wasused to convert voxel count rates to activity concentrations. Theresulting image data were then normalized to the administeredactivity to parameterize images in terms of percent injectedactivity per gram (% IA/g). Manually defined 3-dimensional regionsof interest (also referred to as volumes of interest) were used todetermine the maximum and mean % IA/g (decay corrected to the timeof injection) in various tissues. Images were analyzed using ASIProVM software (Concorde Microsystems).

[0231] Small-Animal CT Imaging and Co-Registration

[0232] Animals that were scanned on both PET and X-ray computedtomography (CT) systems were placed on a custom built platform in arigid body fixed position (using 0.1 mm polyethylene wrapping). Thebed was placed into an integrated heated-air, aneshthesia bed(MultiCell, Mediso). The bed was fixed in place on the microPETgantry and imaged as above. The bed was then moved for CT imagingusing the NanoSPECT/CT (Bioscan). General acquisition parameterswere 55 kVp with a pitch of 1 and 240 projections in a spiral scanmode. The entire animal was scanned using a multiple field of viewprocedure (with an approximate field of view of 4.times.4.times.4cm per bed position), commonly requiring three bed positions perscan. Total scan time was approximately 10 min. A Shepp-Loganfilter was used during the reconstruction process to produce imagematrices with isotropic volumes of 221 .mu.m.

[0233] PET data was reconstructed using a 3D filtered backprojection maximum a priori algorithm using a ramp filter with acut-off frequency equal to the Nyquist frequency into a128.times.128.times.95 matrix. Data was exported in raw format andthe rigid body (3 degrees of freedom) co-registration between PETand CT data (and MR, if applicable) was performed in Amira 5.3.3(FEI). Amira and FIJI was used to produce the majority of thefigures herein.

[0234] Fluorescent Microscopy/Surgical Imaging/ConfocalMicroscopy

[0235] Micrographs were acquired using an Eclipse Ti invertedmicroscope (Nikon) equipped with a motorized stage (PriorScientific Instruments Ltd.), X-cite light source (EXFO) and filtersets (Chroma). Images were acquired and processed usingNIS-Elements (Nikon), FIJI (NIH) and MosaicJ (Phillipe Thevenaz,Biomedical Imaging Group, Swiss Federal Institute of TechnologyLausanne). All fluorescent images were captured with a fixedexposure time (fluorophore dependent).

[0236] Laser scanning confocal microscopy used the TCS SP8 (Leica)in the Molecular Cytology Core Facility (MCCF) of MSKCC. Cells wereplated on glass bottom dishes (NUNC) for 48 hours, washed and thenincubated for the noted time with Cy5.5-IgG (control), Cy5.5-11B6and/or excess blocking 11B6 in supplemented media. Samples werescanned for Cy5.5.

[0237] Cellular Internalization Assay

[0238] VCaP, LNCaP and BT474 (with and without DHT stimulation)cells, cultured according to ATCC guidelines were incubated with.sup.89Zr-11B6 containing media. Uptake mechanism studies usedpurified human non-specific IgG (400 .mu.g/1 mL/well, Invitrogen),human TruStain FcX Fc receptor blocking (40 .mu.L/1 mL/well,Biolegend) or h11B6 (Fab').sub.2 (0.2 mg/l mL/well, DiaProst Corp.)added together with the radioactive antibody. Control wellscontained 20-fold excess of unlabeled antibody (to testspecificity). Antibody concentrations were selected in preliminaryexperiments; a 20-fold increase in the antibody concentration didnot significantly increase the amount of antibody bound. Triplicatesamples were periodically removed, and cells were washed with 1 mLPBS (w/o Ca.sup.2+ and Mg.sup.2++0.2% BSA). Lysate generated (1 mLof 1M NaOH for 5 min) was gamma counted. Cell uptake was determinedby calculating percent activity found in cell lysate [100*(celllysate activity/total activity)].

[0239] Confocal laser scanning microscopy was performed on cellsbeginning 12 h after incubation with 1:200 of either Cy5.5-11B6,phAb-11B6 (Promega Cat. No. G9841) or control Cy5.5-IgG. For FcRnco-localization, cells were fixed, permeabilized and stained usinganti-FcRn Alexa-488 (Fisher, Cat. No. NBP189128-FCGRT).

[0240] Affinity Tests of .sup.89Zr-DFO-11B6, DFO-11B6 andH435A-11B6

[0241] Biotinylated 11B6 (100 .mu.L; 2 mg/L) was added tostreptavidin-coated microtiter plates, followed byl h of incubationwith shaking. The plate was washed, after which 20, 100, 200, 400or 1000 .mu.g of compound (antibody) in 100 .mu.L of DELFIA AssayBuffer was added to the wells, in duplicates, to compete with thecapture antibody. Samples containing 0.34 ng/ml, or 3.4 ng/ml, in100 .mu.L of DELFIA Assay Buffer was hereafter added to the wells.After 2 h incubation with shaking, the plate was washed, and theEu.sup.3+ labeled tracer antibody 6H10 was added (200 .mu.L; 0.5mg/L). The plate was incubated for 1 h with shaking, and thenwashed. DELFIA Enhancement Solution (200 .mu.L) was added, and 5min later, the time-resolved fluorescence was measured.

[0242] Time-Resolved Immunofluorometric Assay of Free and TotalhK2

[0243] Total hK2 was measured using an in-house research assay thathas previously been described by Vaisanen et al. Briefly,streptavidin coated micro-titer plates were incubated withbiotinylated catcher antibody 6H10, followed by washing andincubation with samples and standards. After another round ofwashing, europium labeled tracer antibody 7G1 is added. Afterincubation and washing steps, enhancement solution is added priorto reading the plates. Free hK2 is measured in a similar fashionwith biotin labeled 11B6 as a capture antibody and Europium labeled6H10 as tracer antibody. Both assays have a functional detectionlimit of 0.04 ng/ml.

[0244] Tissue Lysate Preparation and Total Protein Measurement

[0245] Prostate tissues, harvested from transgenic mice werehom*ogenized in lysis buffer (50 mM Sodium Acetate, 2 mM EDTA, 1%Triton X-100, lx complete protease inhibitor (Roche), and 10 mMbenzamidine), sonicated for ten seconds (550 Sonic Dismembrator,Fisher Scientfic) and centrifuged at 13,000 rpm for 10 min. Thesupernatant was saved for analysis of determine free and total hK2levels. Total protein levels were determined in hom*ogenates usingthe BioRad DC Protein assay.

[0246] RNA Isolation and Quantitative-PCR

[0247] Approximately 200 mm.sup.3 of tumor sample was placed in aFastPrep Lysing Matrix tube (MP Biomedicals). Tumors were thenhom*ogenizing in 500 .mu.L of Trizol (Ambion) using a FastPrep-24instrument (MP Biomedicals). For xenograft tumors, the samples weretransferred to a new eppendorf tube where 100 .mu.g of glycogen(Ambion) was added. The samples were mixed by inversion and allowedto sit at room temperature for 5 min. Chloroform (100 .mu.L;OmniSolv) was added and the samples were shaken vigorously andincubated for 3 min. The samples were then centrifuged at 11,500rpm at 4.degree. C. for 15 min and the aqueous (top) phase wastransferred to a new eppendorf tube. Isopropanol (250 .mu.L) of wasadded to the sample by pipetting until a precipitate formed. Thesample was then centrifuged at 11,500 rpm at 4.degree. C. for 10min. The pellet was washed with 75-80% EtOH in DEPC water (Ambion).RNA was then purified using RNeasy Mini Kit (Qiagen) or thePureLink RNA Mini Kit (Ambion). RNA quality and quantity wasdetermined using a spectrophotometer at 260 and 280 nm(Nanodrop-2000, Thermo Scientific). cDNA was generated using theHigh Capacity cDNA Reverse Transcription Kit (Applied Biosystems;Life Technologies). Quantitative-PCR was done using QuantiFast SybrGreen PCR Kit and RT.sup.2 qPCR primers (Qiagen) on aRealPlex.sup.4 Mastercycler system (Eppendorf). KLK2 expression wasquantified relative to beta actin using the comparative CTmethod.

[0248] Single Cell Extractions of Prostatic Tissue

[0249] A suspension of single cells was derived from the excisedmouse prostatic tissue (of animals dosed with 100 .mu.g ofCy5.5-11B6) following mastication at 4.degree. C., digestion for 3h in collagenase/hyaluronidase in culture media (DMEM with 5% FBS)at 37.degree. C., incubation in trypsin for 1 h at 4.degree. C.followed by low speed centrifugation. The cell pellet wasresuspended in 5 mg/mL dispase and 1 mg/mL DNase I and pipettedgently, before being passed through a 70 .mu.m strainer(ThermoFisher). All reagents were purchased from Stem CellTechnologies unless otherwise noted. Aliquots of the suspensionwere placed between two glass coverslips and scanned on the EclipseTi, as above.

[0250] Statistical Analysis

[0251] Data are presented as means.+-.standard of the mean, unlessotherwise noted. Statistical significance was analyzed bynonparametric student t test. Pearson's correlation coefficientswere used for assessing the strength of association between pairsof predefined variables. In all cases, differences in results wereconsidered to be statistically significant when the computed Pvalue was less than 0.05. All tests were two-tailed. Analyses wereperformed using Prism 6.0 (Graphpad).

[0252] Expression and Purification of hu11B6

[0253] HEK293 cells were expanded to a cell density of1.times.10.sup.6 cells/mL in a 2 L suspension culture in FreeStyle293 Expression Medium (Life Technologies). The plasmid DNA(expression vectors p11B6VLhV1hk and p11B6VHhV1hIgG.sub.1)containing the nucleotide sequences for the heavy and light chainsof hu11B6 IgG1/k was then mixed with the transfection agent andincubated for 10 min at room temperature (RT). The DNA transfectionagent mix was slowly added to the cell culture while slowlyswirling the flask. The transfected cell culture was then incubatedat 37.degree. C. with 8% CO.sub.2 on an orbital shaker platformrotating atabout 135 rpm for seven days. Culture medium washarvested by centrifugation and filtered through 5 .mu.m, 0.6.mu.m, and 0.22 .mu.m filter systems. Antibodies were purified byProtein G chromatography, and the buffer was changed to PBS pH 7.4by dialysis; subsequently, the antibodies were concentrated byultrafiltration. Concentration was measured by absorbance. Overallyield was 13.1 mg (.about.6.5 mg/L).

[0254] Tissue Histology and Autoradiography

[0255] After mice were euthanized, a tissue package containingprostate lobes, seminal vesicles, and prostatic urethra wassurgically excised and incubated in Tissue-Tek optimal cuttingtemperature compound (Sakura Finetek USA, Inc.) on ice for 45minutes, and then snap-frozen on dry ice in a cryomold. Sets ofcontiguous 15 or 100 .mu.m-thick tissue sections were cut with aCM1950 cryostat microtome (Leica Microsystems Inc.) and arrayedonto SuperfrostPlus glass microscope slides. Sections stained foractin and DNA (100 .mu.m sections) were incubated with 200 .mu.L of10 U/mL rhodamine-phalloidin (Life Sciences Inc.) in PBS for 2-3hours at RT in a covered container to prevent evaporation, and thenwashed with PBS twice. DNA/nuclei staining was performed byincubating the slides for 10 min in 5 .mu.g/mL DAPI in PBS,followed by a wash with PBS. Slides were then air-dried, and a dropof Mowiol A-48 (Calbiochem Inc.) was placed on the slide beforeadding a mounting cover glass. Slides were then stored at-20.degree. C. Immunostaining for AR was performed by incubatingslides with blocking solution (2% BSA in PBS) for 15 min at roomtemperature and staining with 1:200 dilution of anti-AR polyclonalantibody (NH27) for 45 min followed by Texas red-conjugated goatanti-rabbit antibody (ICN) for 45 min at room temperature. Stainedslides were then washed and mounted.

[0256] Sections intended for autoradiography were fixed in 4%paraformaldehyde solution in phosphate-buffered saline (Affymetrix)for 5 minutes, washed twice, air-dried, and stained withhematoxylin and eosin (H&E). The immunohistochemical detectionof Ki-67, AR (N-20), and c-MYC was performed at the MolecularCytology Core Facility of Memorial Sloan Kettering Cancer Centerusing a Discovery XT processor (Ventana Medical Systems). Beforestaining, all sections were blocked for 30 minutes in 10% normalgoat serum with 2% BSA in PBS. Sections stained for Ki-67 wereincubated with 0.4 .mu.g/mL of the primary antibody (rabbitpolyclonal Ki-67 antibody; Vector Labs, cat.#: VP-K451) for 2hours, followed by a 30-minute incubation with biotinylated goatanti-rabbit IgG (Vector Labs, cat.#:PK6101) at 1:200 dilution.Sections stained for AR (N-20) were incubated for 3 hours with apolyclonal rabbit antibody (Santa Cruz, cat.#: SC-816) at 1.mu.g/ml concentration, followed by 16 minutes of incubation withbiotinylated goat anti-rabbit IgG (Vector labs, cat#:PK6101) at1:200 dilution. C-MYC staining was performed by incubating sectionsfor 5 hours with a primary anti-c-MYC antibody (N terminal, rabbitpolyclonal, Epitomics, cat.#: P01106), followed by 60 minutes ofincubation with biotinylated goat anti-rabbit IgG (Vector Labs,cat.#: PK6101) at 1:200 dilution. Blocker D, streptavidin-HRP, andDAB detection kit (Ventana Medical Systems) were used according tothe manufacturer's instructions. Stained tissue sections wereplaced in a film cassette against a Fuji film BAS-MS2325 imagingplate (Fuji Photo Film Co.) to acquire digital autoradiograms. Theslides were exposed for 48 hours, approximately 168 hours afterinjection of .sup.89Zr-DFO-11B6. Exposed phosphor plates were readby a Fujifilm BAS-180011 bio-imaging analyzer (Fuji Photo FilmCo.), generating digital images with 50 .mu.m pixel resolution.Digital images were obtained with an Olympus BX60 System Microscope(Olympus America, Inc.) equipped with a motorized stage (PriorScientific, Inc.). Subsequently, H&E images were acquired tothe same resolution as the DAR data. DAR images were manuallyaligned to the H&E images using rigid planar transforms.

[0257] Transgenic KLK2 Mouse Models

[0258] Site-directed mutagenesis of APLILSR to APLRTKR at positions4, -3, and -2 the zymogen sequence of KLK2 was performed using aQuick Change Lightning Mutagenesis Kit (Stratagene). This enabledfurin, a ubiquitously expressed protease in rodent prostate tissue,to efficiently cleave the short activation peptide at the cleavagesite (-1 Arg/+1 Ile), resulting in functional hK2. Sequencing wasperformed to verify the genotype using the following primers:5'-TTC TCT AGG CGC CGG AAT TA-3' (forward), 3'-CCC GGT AGA ATT CGTTAA CCT-3' (reverse). A transgenic mouse model was established bycloning the described construct into a SV40 T-antigen cassettedownstream of the short rat probasin promoter (pb). This constructwas microinjected into fertilized mouse embryos (C57BL/6) andimplanted into pseudopregnant female mice. A cancer-susceptibletransgenic mouse model with prostate specific hK2 expression wascreated by crossing the pb_KLK2 transgenic model with the Hi-MYCmodel (ARR2PB-Flag-MYC-PAI transgene). A schematic of thestrategies used is included as FIG. 31. Integration of genes intothe genome of the offspring was confirmed by Southern blot analysisand PCR. Mice were monitored closely in accordance withIACUC-established guidelines and RARC animal protocol(#04-01-002).

[0259] Castration- and Enzalutamide-Resistant Liver MetastasisModel

[0260] Previously surgically castrated mice with a body weight of28-30 g were anesthetized by intraperitoneal injection of ketamine(75 mg/kg) and xylazine 2% (15 mg/kg). Anesthetized animals wereplaced in a supine position, draped, and prepared for sterilesurgery. A 10 mm midline incision was made on the upper abdomenthrough the skin and peritoneum. The left lobe of the liver wasseparated from the caudate and median lobe, and was exposed andimmobilized. A Hamilton syringe with a 26-gauge needle was used forinjection of a 10 mixture of LREX' tumor cells (10.sup.5 cells) andMatrigel (1:1). The puncture site was closed by gentle pressure forapproximately 1 min with a moistened cotton-tipped applicatorstick. After tumor cell inoculation, the liver lobe wasrepositioned anatomically. The abdominal wall was then closed in atwo-layer technique with a resorbable suture for the fascia andsubcutaneous tissue (5/0 vicryl, Ethicon) and a nonresorbablesuture for the skin (5/0 prolene, Ethicon). A 0.05 mg dexamethasonepellet (60 day release) was subcutaneously implanted at the end ofthe procedure to confer enzalutamide resistance and activate theglutocorticoid receptor (47). Animals received postoperativeanalgesia by subcutaneous injection of carprofen (5 mg/kg) oncedaily for 3 days after surgery. Daily enzalutamide (10 mg/kg)treatment was given by gavage. Tumor development was followed withbioluminescence imaging and confirmed with MR imaging.

[0261] Antibody Humanization

[0262] The acceptor framework used for the grafting was derivedfrom the human immunoglobulin germline genes showing the highestsequence similarity with the variable domains of the parental 11B6antibody. The genes were identified by comparing the amino acidsequences of the mouse 11B6 variable light (V.sub.L) and heavy(V.sub.H) domains to the human immunoglobulin germline sequences inNCBI database. The germline V gene IGKV4-1*01 (GenBank: Z00023.1)together with the short IGKJ2 gene (GenBank: J00242.1) wereselected to construct the V.sub.L acceptor framework into which theCDRs of mouse 11B6 light chain were grafted. For the V.sub.Hacceptor framework, the V gene IGHV4-28*01 (GenBank: X05714.1) andJ gene IGHJ1 (GenBank: AAB59411.1) were used. A 3D hom*ology modelof the mouse 11B6 was built to facilitate the evaluation of theinfluence of non-CDR residues on the CDR loop conformations. On thebasis of the published data and visual inspection of the model, thefollowing residues were adopted from the parental mouse 11B6: Leu4in the light chain and Asn27, Thr30, Arg71, and Thr94 in the heavychain. On the basis of structural analysis, certain CDR residueswere obtained from the sequences of the human acceptor framework:an arginine was introduced in the position 54 in CDR-L2 to allowthe formation of a salt bridge with another light chain residueAsp60, whereas Lys24 in CDR-L1 and Asn60 in CDR-H2 were included tomaximize the content of human gene-derived amino acids in hu11B6,although they were predicted not to play a major role in antigenbinding.

[0263] Codon optimized nucleotide sequences encoding hu11B6variable heavy or light chains were designed, purchased assynthetic genes, and subcloned to obtain the mammalian expressionvectors p11B6VLhV1hk (4300 bp) and p11B6VHhV1hIgG.sub.1 (4900 bp)for the production human IgG.sub.1/kappa antibody.

[0264] 11B6 Immunohistochemistry

[0265] The murine 11B6 antibody was used on human tissuemicroarrays. Human tissue microarrays (US Biomax) included fineneedle biopsies of normal prostate, primary adenocarcinoma, andmetastatic foci. Four-.mu.m sections were deparrafinized in xyleneand rehydrated in decreasing ethanol dilutions. Endogenousperoxidase was blocked with 3% hydrogen peroxide buffer for 10minutes. Antigen retrieval was performed by boiling in EDTA buffer(pH 9.0) for 20 min. Slides were subsequently incubated overnightin a humidified chamber with murine anti-hK2 (m11B6) at a 1:1000dilution in 0.5% BSA/TBST followed by one hour incubation withPoly-HRP-anti-mouse/rabbit/rat IgG (Brightvision, Immunologic). Theslides were developed with diaminobenzidine and lightlycounterstained with hematoxylin and mounted.

[0266] FcRn Affinity Measurements

[0267] To test the effect of the H435A-11B6 antibody, whichcontains a point mutation (and the original 11B6 construct),surface plasmon resonance (SPR) was performed on a CMS chip using aBiacore 3000 instrument. The chip and all reagents were purchasedfrom GE Healthcare; experiments were conducted in assay buffer (67mM phosphate buffer, 0.15 M NaCl, 0.05% Tween-20) adjusted toeither pH 6.0 or pH 7.4. At the lower pH, FcRn has the ability tobind to the Fc portion of intact immunoglobulins (IgG.sub.1), butat the higher pH this affinity drops to enable release of theantibody (24). Human FcRn (hFcRn) was bound to the chip byfollowing the manufacturer's guidelines, with carbodiimide (EDC)and N-hydroxysuccinimide (NETS) in reaction buffer (10 mM sodiumacetate, pH 5.0) and washed after immobilization with runningbuffer. Channels were blocked by ethanolamine after activation andimmobilization and EDC and NETS washed off. The affinity of eachantibody for the FcRn was evaluated with a flow rate of 30.mu.L/min at a concentration of 50 nM in each buffer condition. Ifbinding was observed, association and dissociation rates weremeasured using the bivalent fitting model (BIAevaluation Software,Biacore).

[0268] Characterization of h11B6 Affinity

[0269] After optimizing the experimental conditions, multiplebinding measurements were performed for m11B6, hu11B6, DFO-hu11B6,and the antigen. From the collected data, the association anddissociation rate constants (k.sub.on and k.sub.off) and thedissociation constants (K.sub.D) were calculated.

EQUIVALENTS

[0270] While systems, methods, and compositions have beenparticularly shown and described with reference to specificpreferred embodiments, it should be understood by those skilled inthe art that various changes in form and detail may be made thereinwithout departing from the spirit and scope of the invention asdefined by the appended claims.

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References

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