Anti-PSMA x Anti-CD3 Bispecific Antibody Redirects T Cell ...
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Anti-PSMA x Anti-CD3 Bispecific Antibody Redirects T Cell Cytotoxicity in Castrate-Resistant Prostate Cancer Models Toddy Sewell, Ruth A. Chenault, Jennifer Wiens, John Kumer, Robert Bader, Sateesh Natarajan, Catherine J. McMahan, Paul A. Algate, and John W. Blankenship Emergent Product Development Seattle – Seattle, WA, USA Introduction Prostate cancer is the most common cancer in men. Although screening, radiation, surgery and hormone ablation therapy have greatly improved the detection and treatment of early stage prostate cancer, few options exist to treat metastatic, castrate-resistant prostate cancer (CRPC). We have developed a therapeutic approach that uses a bispecific SCORPION™ (multi-specific protein therapeutic) molecule that redirects T cell cytotoxicity against cells expressing a common prostate cancer antigen, PSMA (Prostate Specific Membrane Antigen). Targeting PSMA Figure 1. PSMA Structure Image created using PyMOL (www.pymol.org). PDB ID: 2OOT PSMA, also known as folate hydrolase 1 (FOLH1), glutamate carboxypeptidase II (GCPII), and N-acetyl-α-linked acidic dipeptidase I (NAALDase), is a dimeric cell membrane-bound enzyme involved in folate metabolism, which is most commonly found in prostate epithelial cells. PSMA was selected as the target tumor antigen because of its close association with prostate cancer. 1-2 Briefly, in prostate cancer cells, PSMA is expressed 1,000-fold higher than on normal prostate epithelium. 3 Expression of PSMA increases with prostate cancer pro- gression and is highest in metastatic disease, hormone refractory disease, and higher-grade lesions. 4-8 Relative PSMA expression within clinical samples has also been shown to correlate with overall disease outcome for prostate cancer patients. 9 SCORPION Therapeutics SCORPION molecules are bispecific antibody-like therapeutics containing two sets of binding domains linked to immunoglobulin Fc domains to extend the half-life of the molecule in vivo. In the αPSMA x αCD3 SCORPION molecule, the Fc region has been engineered to minimize complement fixation and interaction with Fcγ receptors, to avoid interactions with other components of the immune system that could lead to CD3 clustering and non-specific T cell activation. The anti-PSMA binding domain is a humanized single chain variable fragment (scFv) derived from a murine antibody that binds human and non- human primate (NHP) PSMA. The anti-CD3 binding domain is a humanized scFv adapted from a murine antibody that binds human and NHP CD3. Figure 2. Antibody αPSMA x αCD3 SCORPION Molecule αPSMA x αCD3 Molecular Model Redirected T Cell Cytotoxicity Figure 3. Multiple bispecific antibody formats have been shown to redirect T cell cyto- toxicity by physically cross-linking the T cell receptor complex to a cell surface antigen on target cells. This drives formation of a cytotoxic synapse and triggers T cell activation and tumor cell lysis in a target antigen-dependent fashion. 10-11 Binding Studies Binding of both ends of the αPSMA x αCD3 SCORPION molecule was assessed by flow cytometry on PSMA and CD3ε expressing cell lines. Binding to recombinant human and non-human primate PSMA ectodomains was tested by ELISA. Figure 4. Binding to PSMA on Tumor Cells (flow cytometry) Binding to Human PSMA ECD (ELISA) Binding to CD3 on Junkat Cells (flow cytometry) Binding to NHP PSMA ECD (ELISA) Target Method EC 50 Human PSMA (C4-2B) Flow 4.0 ± 0.4 nM Human PSMA (MDA-PCa-2b)* Flow 9.5 ± 1.0 nM Human CD3ε (Jurkat) Flow 2.7 ± 0.15 nM Human PSMA ECD ELISA 5.8 ± 0.2 nM Rhesus PSMA ECD ELISA 11 ± 2.4 nM *Data not shown In Vitro Cytotoxicity C4-2B and MDA-PCa-2b (both PSMA+/RON-/CD19-) target cells were loaded with 51 Cr and incubated with SCORPION protein and primary human or cyno- molgus monkey T cells for 4 or 24 hours. The percentage of target cell lysis was measured by specific 51 Cr release into the supernatant as compared to total target cell lysis with NP-40. SCORPION molecules specific to other tumor antigens (RON, CD19) were added as negative controls. Figure 5. Human T Cells, 24 h Cyno T Cells, 4 h Target T Cells Time EC 50 C4-2B Human 24 h 4.2 ± 0.47 pM MDA-PCa-2b Human 24 h 30 ± 16 pM C4-2B Cyno 4 h 30 ± 5 pM In Vitro T Cell Activation/Proliferation Figure 6. CD4 + T Cells CD4 + T Cells CD8 + T Cells CD8 + T Cells Purified human T cells were labeled with CFSE and incubated for 4 days at 37°C with SCORPION protein alone or with SCORPION protein and either C4- 2B (PSMA+) target or MDA-MB-453 (PSMA-) non-target cells rendered non- proliferative by Mitomycin C treatment. The ratio of target cells to T cells was 1:3. Data is plotted as the percent of T cells that were activated, as assessed by CD25 staining, or proliferated, as assessed by loss of CFSE fluorescence. Stability in Mouse Serum Figure 7. Cytotoxicity. Human T Cells, C4-2B Target Cells, 24 h. Condition Time Assay Time EC 50 37ºC Human 24 h 15 ± 3.1 pM 4ºC Human 24 h 10 ± 2.2 pM -20ºC Cyno 4 h 11 ± 1.9 pM Serum stability was assessed by incubating αPSMA x αCD3 SCORPION protein in mouse serum and performing a cytotoxicity assay as described above. In Vivo Efficacy Model Figure 8. Tumor Volumes, C4-2B Prophylactic Efficacy Model (last recorded volume carried forward after endpoint reached) 2 million C4-2B cells were mixed with 1 million purified human T cells in solubilized basement membrane matrix and injected subcutaneously into the flank of male NOD/SCID mice. SCORPION molecules were administered by IV injection on days 0, 4, and 8. Negative controls included treatment with vehicle alone or treatment with SCORPION molecule in the absence of T cells. Animals were monitored for tumor growth and weight loss endpoint criteria. Tumor volumes are plotted as the mean with the standard error; points are plotted as solid colors until the first mouse in each group reaches an tumor growth or weight loss endpoint. Figure 9. Percent Survival, C4-2B Prophylactic Efficacy Model Statistics assessed by Log-rank (Mantel-Cox) Test comparison performed between “T cells + Vehicle” control and individual treatment groups. All treat - ment groups showed a statistically significant increase in survival, compared to negative controls. Summary and Conclusions — SCORPION molecules targeting PSMA and CD3 redirected T-cell cytotoxicity against PSMA(+) cell lines in vitro at low picomolar concentrations. — Cytotoxic activity was target dependent and required the presence of T cells. — T cells were activated and proliferated in response to the SCORPION therapeutic in the presence of PSMA(+) target cells; no T cell activation or proliferation was observed in the absence of target cells. — SCORPION proteins were stable after freeze/thaw in mouse serum and showed stable functional activity in mouse serum. — Treatment with anti-PSMA x anti-CD3 SCORPION protein inhibited the growth of subcutaneous tumors in the presence of human T cells in a mouse xenograft model of prostate cancer. All treatment groups showed a statistically significant increase of survival compared to negative controls. Conclusions: These studies show that anti-PSMA x anti-CD3 SCORPION therapeutics redirect T cell cytotoxicity and show promise as potential thera- peutics in CRPC. References 1. O’Keefe DS, et al (2001). “Prostate Specific Membrane Antigen”. in Simons JW, Chung LWK, Isaacs WB. Prostate cancer: biology, genetics and the new therapeutics. Totowa, NJ: Humana Press. pp. 307–326. 2. Wolf, P. (2011) “Chapter 4: Prostate Specific Membrane Antigen as Biomarker and Therapeutic Target for Prostate Cancer.”, in Spiess PE, Prostate Cancer – Diagnostic and Therapeutic Advances, InTech. pp 81-100. 3. Su LS, et al. Alternatively Spliced Variants of Prostate-specific Membrane Antigen RNA: Ratio of Expression as a Potential Measurement of Progression. Cancer Research (1995) 44, 1441. 4. Wright GL, et al. Expression of prostate-specific membrane antigen in normal, benign, and malignant prostate tissues. Urologic Oncology: Seminars and Original Investigations. (1995) 1, 18. 5. Israeli RS, et al. Expression of the Prostate-specific Membrane Antigen. Cancer Res (1994) 54, 1807. 6. Sweat SD, et al. Prostate-specific membrane antigen expression is greatest in prostate adenocarcinoma and lymph node metastases. Urology (1998) 52, 637. 7. Lapidus, R.G. et al. Prostate-specific membrane antigen (PSMA) enzyme activity is elevated in prostate cancer cells. Prostate (2000) 45(4), 350. 8. Burger, M.J. et al Expression analysis of delta-catenin and prostate-specific membrane- antigen: their potential as diagnostic markers for prostate cancer. Int J Cancer (2002) 100(2), 228. 9. Ross JS et al, Correlation of primary tumor prostate-specific membrane antigen expression with disease recurrence in prostate cancer. Clin Cancer Res (2003) 9(17), 6357. 10. Dreier T, et al. Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody. Int. J. Cancer (2002) 100, 690. 11. Wolf E, et al. BiTEs: bispecific antibody constructs with unique anti-tumor activity. Drug Discovery Today (2005) 10, 1237. SCORPION™ and any and all Emergent BioSolutions, Inc. brand, product, service and feature names, logos, and slogans are trademarks or registered trademarks of Emergent BioSolutions, Inc. or its subsidiaries in the United States or other countries. All rights reserved. EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics: November 6-9, 2012 in Dublin, Ireland Image created using Accelrys Discovery Studio and PyMOL (www.pymol.org). 0 50 100 150 0.1 1 10 100 αPSMAxαCD3 on C4-2B cells (PSMA+) αPSMAxαCD3 on DU-145 cells (PSMA-) Concentration (nM) MFI Concentration (nM) MFI 0.1 1 10 100 0 500 1000 1500 αPSMAxαCD3 on Jurkat cells (CD3+) 0 10000 20000 30000 40000 0.001 0.01 0.1 1 10 100 Concentration (nM) RLU 0 500 1000 1500 0.001 0.01 0.1 1 10 100 Concentration (nM) RLU αPSMAxαCD3 αPSMAxαCD3 Concentration (pM) % Specific Lysis 0.1 1 10 100 1000 10000 0 10 20 30 40 αPSMA x αCD3 on C4-2B cells αRON x αCD3 on C4-2B cells (off-target) Concentration (pM) % Specific Lysis 0.1 1 10 100 0 20 40 60 80 α PSMA x α CD3 (C4-2B) α CD19 x α CD3 (C4-2B) (off-target) α PSMA x α CD3 (MDA-PCa-2b) α CD19 x α CD3 (MDA-PCa-2b) (off-target) 0 20 40 60 80 100 0.1 1 10 100 1000 10000 0 Concentration (pM) % CD25 + 0 20 40 60 80 100 0.1 1 10 100 1000 10000 0 Concentration (pM) % CD25 + Activation 0 20 40 60 80 100 0.1 1 10 100 1000 10000 0 Concentration (pM) % Dividing Proliferation 0 20 40 60 80 100 0.1 1 10 100 1000 10000 0 Concentration (pM) % Dividing αPSMA x αCD3 on T cells alone αPSMA x αCD3 on C4-2B αPSMA x αCD3 on MDA-MB-453 0.1 0.3 1 3.2 10 31.6 100 316.2 0 20 40 60 Concentration (pM) % Specific Lysis 7 d at -20 C 0 d at 37 C 7 d at 4 C Treatment Groups (n=15): Treatment Day 0, 4, 8 Study Day Ave. Tumor Volume (mm 3 ) 0 25 50 75 100 125 0 250 500 750 1000 1250 1500 T cells + 30 ug αPSMA x αCD3 T cells + 0.3 ug αPSMA x αCD3 T cells + 3 ug αPSMA x αCD3 T cells + Vehicle (true control) Matrigel only (no tumor cells) Vehicle (Tumor only, no T cells) 30 ug αPSMA x αCD3 (no T cells) Study Day Percent survival 0 25 50 75 100 125 0 20 40 60 80 100 T cells + 30 ug αPSMA x αCD3 *** T cells + 0.3 ug αPSMA x αCD3 ** T cells + 3 ug αPSMA x αCD3 ** T cells + Vehicle (true control) p<0.05*, p<0.01**, p<0.001*** Vehicle (Tumor only, no T cells) 30 ug αPSMA x αCD3 (no T cells) Treatment Groups (n=15): T cell cytotoxic granule TCR complex PSMA Bispecific links tumor cell and T cell Tumor cell Formation of cytolytic synapse Lysis of tumor cell T cell activation and proliferation CH3 CH3 CH2 CH2 CH1 VH1 CL VL1 CH1 VH1 CL VL1 ADCC and CDC Functional VH2 VL2 VH2 VL2 VH1 VL1 VH1 VL1 CH3 CH3 CH2 CH2 Anti-PSMA Anti-CD3 IgG ADCC and CDC null (stable Ab tail with no effector function)
Transcript of Anti-PSMA x Anti-CD3 Bispecific Antibody Redirects T Cell ...
Anti-PSMA x Anti-CD3 Bispecific Antibody Redirects T Cell Cytotoxicity in Castrate-Resistant Prostate Cancer ModelsToddy Sewell, Ruth A. Chenault, Jennifer Wiens, John Kumer, Robert Bader, Sateesh Natarajan, Catherine J. McMahan, Paul A. Algate, and John W. Blankenship Emergent Product Development Seattle – Seattle, WA, USA
Introduction
Prostate cancer is the most common cancer in men. Although screening, radiation, surgery and hormone ablation therapy have greatly improved the detection and treatment of early stage prostate cancer, few options exist to treat metastatic, castrate-resistant prostate cancer (CRPC). We have developed a therapeutic approach that uses a bispecific SCORPION™ (multi-specific protein therapeutic) molecule that redirects T cell cytotoxicity against cells expressing a common prostate cancer antigen, PSMA (Prostate Specific Membrane Antigen).
Targeting PSMA
Figure 1. PSMA Structure
Image created using PyMOL (www.pymol.org). PDB ID: 2OOT
PSMA, also known as folate hydrolase 1 (FOLH1), glutamate carboxypeptidase II (GCPII), and N-acetyl-α-linked acidic dipeptidase I (NAALDase), is a dimeric cell membrane-bound enzyme involved in folate metabolism, which is most commonly found in prostate epithelial cells. PSMA was selected as the target tumor antigen because of its close association with prostate cancer.1-2 Briefly, in prostate cancer cells, PSMA is expressed 1,000-fold higher than on normal prostate epithelium.3 Expression of PSMA increases with prostate cancer pro-gression and is highest in metastatic disease, hormone refractory disease, and higher-grade lesions.4-8 Relative PSMA expression within clinical samples has also been shown to correlate with overall disease outcome for prostate cancer patients.9
SCORPION Therapeutics
SCORPION molecules are bispecific antibody-like therapeutics containing two sets of binding domains linked to immunoglobulin Fc domains to extend the half-life of the molecule in vivo. In the αPSMA x αCD3 SCORPION molecule, the Fc region has been engineered to minimize complement fixation and interaction with Fcγ receptors, to avoid interactions with other components of the immune system that could lead to CD3 clustering and non-specific T cell activation. The anti-PSMA binding domain is a humanized single chain variable fragment (scFv) derived from a murine antibody that binds human and non- human primate (NHP) PSMA. The anti-CD3 binding domain is a humanized scFv adapted from a murine antibody that binds human and NHP CD3.
Figure 2.
Antibody αPSMA x αCD3 SCORPION Molecule
αPSMA x αCD3 Molecular Model
Redirected T Cell Cytotoxicity
Figure 3.
Multiple bispecific antibody formats have been shown to redirect T cell cyto-toxicity by physically cross-linking the T cell receptor complex to a cell surface antigen on target cells. This drives formation of a cytotoxic synapse and triggers T cell activation and tumor cell lysis in a target antigen-dependent fashion.10-11
Binding Studies
Binding of both ends of the αPSMA x αCD3 SCORPION molecule was assessed by flow cytometry on PSMA and CD3ε expressing cell lines. Binding to recombinant human and non-human primate PSMA ectodomains was tested by ELISA.
Figure 4.
Binding to PSMA on Tumor Cells (flow cytometry)
Binding to Human PSMA ECD (ELISA)
Binding to CD3 on Junkat Cells (flow cytometry)
Binding to NHP PSMA ECD (ELISA)
Target Method EC50
Human PSMA (C4-2B) Flow 4.0 ± 0.4 nM
Human PSMA (MDA-PCa-2b)* Flow 9.5 ± 1.0 nM
Human CD3ε (Jurkat) Flow 2.7 ± 0.15 nM
Human PSMA ECD ELISA 5.8 ± 0.2 nM
Rhesus PSMA ECD ELISA 11 ± 2.4 nM*Data not shown
In Vitro Cytotoxicity
C4-2B and MDA-PCa-2b (both PSMA+/RON-/CD19-) target cells were loaded with 51Cr and incubated with SCORPION protein and primary human or cyno-molgus monkey T cells for 4 or 24 hours. The percentage of target cell lysis was measured by specific 51Cr release into the supernatant as compared to total target cell lysis with NP-40. SCORPION molecules specific to other tumor antigens (RON, CD19) were added as negative controls.
Figure 5.
Human T Cells, 24 h Cyno T Cells, 4 h
Target T Cells Time EC50
C4-2B Human 24 h 4.2 ± 0.47 pM
MDA-PCa-2b Human 24 h 30 ± 16 pM
C4-2B Cyno 4 h 30 ± 5 pM
In Vitro T Cell Activation/Proliferation
Figure 6.
CD4+ T Cells
CD4+ T Cells
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Purified human T cells were labeled with CFSE and incubated for 4 days at 37°C with SCORPION protein alone or with SCORPION protein and either C4-2B (PSMA+) target or MDA-MB-453 (PSMA-) non-target cells rendered non- proliferative by Mitomycin C treatment. The ratio of target cells to T cells was 1:3. Data is plotted as the percent of T cells that were activated, as assessed by CD25 staining, or proliferated, as assessed by loss of CFSE fluorescence.
Stability in Mouse Serum
Figure 7. Cytotoxicity. Human T Cells, C4-2B Target Cells, 24 h.
Condition Time Assay Time
EC50
37ºC Human 24 h 15 ± 3.1 pM
4ºC Human 24 h 10 ± 2.2 pM
-20ºC Cyno 4 h 11 ± 1.9 pM
Serum stability was assessed by incubating αPSMA x αCD3 SCORPION protein in mouse serum and performing a cytotoxicity assay as described above.
In Vivo Efficacy Model
Figure 8. Tumor Volumes, C4-2B Prophylactic Efficacy Model (last recorded volume carried forward after endpoint reached)
2 million C4-2B cells were mixed with 1 million purified human T cells in solubilized basement membrane matrix and injected subcutaneously into the flank of male NOD/SCID mice. SCORPION molecules were administered by IV injection on days 0, 4, and 8. Negative controls included treatment with vehicle alone or treatment with SCORPION molecule in the absence of T cells. Animals were monitored for tumor growth and weight loss endpoint criteria. Tumor volumes are plotted as the mean with the standard error; points are plotted as solid colors until the first mouse in each group reaches an tumor growth or weight loss endpoint.
Figure 9. Percent Survival, C4-2B Prophylactic Efficacy Model
Statistics assessed by Log-rank (Mantel-Cox) Test comparison performed between “T cells + Vehicle” control and individual treatment groups. All treat-ment groups showed a statistically significant increase in survival, compared to negative controls.
Summary and Conclusions
— SCORPION molecules targeting PSMA and CD3 redirected T-cell cytotoxicity against PSMA(+) cell lines in vitro at low picomolar concentrations.
— Cytotoxic activity was target dependent and required the presence of T cells.
— T cells were activated and proliferated in response to the SCORPION therapeutic in the presence of PSMA(+) target cells; no T cell activation or proliferation was observed in the absence of target cells.
— SCORPION proteins were stable after freeze/thaw in mouse serum and showed stable functional activity in mouse serum.
— Treatment with anti-PSMA x anti-CD3 SCORPION protein inhibited the growth of subcutaneous tumors in the presence of human T cells in a mouse xenograft model of prostate cancer. All treatment groups showed a statistically significant increase of survival compared to negative controls.
Conclusions: These studies show that anti-PSMA x anti-CD3 SCORPION therapeutics redirect T cell cytotoxicity and show promise as potential thera-peutics in CRPC.
References
1. O’Keefe DS, et al (2001). “Prostate Specific Membrane Antigen”. in Simons JW, Chung LWK, Isaacs WB. Prostate cancer: biology, genetics and the new therapeutics. Totowa, NJ: Humana Press. pp. 307–326.
2. Wolf, P. (2011) “Chapter 4: Prostate Specific Membrane Antigen as Biomarker and Therapeutic Target for Prostate Cancer.”, in Spiess PE, Prostate Cancer – Diagnostic and Therapeutic Advances, InTech. pp 81-100.
3. Su LS, et al. Alternatively Spliced Variants of Prostate-specific Membrane Antigen RNA: Ratio of Expression as a Potential Measurement of Progression. Cancer Research (1995) 44, 1441.
4. Wright GL, et al. Expression of prostate-specific membrane antigen in normal, benign, and malignant prostate tissues. Urologic Oncology: Seminars and Original Investigations. (1995) 1, 18.
5. Israeli RS, et al. Expression of the Prostate-specific Membrane Antigen. Cancer Res (1994) 54, 1807.
6. Sweat SD, et al. Prostate-specific membrane antigen expression is greatest in prostate adenocarcinoma and lymph node metastases. Urology (1998) 52, 637.
7. Lapidus, R.G. et al. Prostate-specific membrane antigen (PSMA) enzyme activity is elevated in prostate cancer cells. Prostate (2000) 45(4), 350.
8. Burger, M.J. et al Expression analysis of delta-catenin and prostate-specific membrane-antigen: their potential as diagnostic markers for prostate cancer. Int J Cancer (2002) 100(2), 228.
9. Ross JS et al, Correlation of primary tumor prostate-specific membrane antigen expression with disease recurrence in prostate cancer. Clin Cancer Res (2003) 9(17), 6357.
10. Dreier T, et al. Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody. Int. J. Cancer (2002) 100, 690.
11. Wolf E, et al. BiTEs: bispecific antibody constructs with unique anti-tumor activity. Drug Discovery Today (2005) 10, 1237.
SCORPION™ and any and all Emergent BioSolutions, Inc. brand, product, service and feature names, logos, and slogans are trademarks or registered trademarks of Emergent BioSolutions, Inc. or its subsidiaries in the United States or other countries. All rights reserved.
EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics: November 6-9, 2012 in Dublin, Ireland
Image created using Accelrys Discovery Studio and PyMOL (www.pymol.org).
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