anti-CD3 Activated T Cells Research Articles

A Novel Combination of Anti-CD3 x Anti-CD123 Monoclonal Antibodies to Redirect Activated T Cells to Target Acute Myelogenous Leukemia

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy of the bone marrow (BM) and blood, characterized by clonal expansion of neoplastic myeloid precursor cells, and impairment of normal hematopoiesis. AML accounts for the largest number of annual deaths from leukemias among adults in the United States. AML blasts as well as leukemic stem cells express high levels of interlekin-3 (IL-3) receptor-a(CD123). Our previous studies show the anti-CD3 activated T cells (ATC) can be redirected with bispecific antibodies (BiAb) to CD20 for non-Hodgkin's lymphoma after stem cell transplantation (SCT), Her2/ neu for breast cancer, EGFR for pancreatic cancer and GD2 for neuroblastoma. In this study, we investigated whether ATC armed with anti-CD3 x anti-CD123 bispecific antibody (CD123Bi) can target CD123 expressing targets. Methods: ATC were produced by stimulating peripheral blood mononuclear cells (PBMC) with anti-CD3 in the presence of IL-2. Bispecific antibody was produced by chemical hetero-conjugation of anti-CD3 (OKT3) with anti-CD123 monoclonal antibodies. We tested four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) as targets for cytotoxicity by CD123Bi armed ATC using 51Cr release assay. Cytokines and chemokine release during CD123Bi armed ATC mediated killing of targets were analyzed by multiplex luminex assay. Results: All four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) showed 70-90% CD123 expression and 10-70% CD123 expression on leukemic blasts. Arming dose titration ranging from 0.1 to 100 ng CD123Bi/106 ATC showed saturation at 5 ng dose, the mean specific cytotoxicity 20% plateaued at arming doses of 5, 50 and 100 ng/106 ATC at an effector to target ratio (E/T) of 10:1. ATC from 4 normal donors at 50 ng arming dose at E/T of 25:1 showed mean specific cytotoxicity of 22 and 40% directed at TF-1 and NoMo-1 cell lines, respectively. The cytotoxicity was also confirmed by analysis of IFNγ and TNFα release during cytotoxic activity directed at AML cell lines by CD123Bi armed ATC. Conclusions: ATC armed with CD123Bi at 50 ng/106 cells showed comparable cytotoxicity directed at either high or low leukemic blast populations. Since most of the CD33 positive cells were also CD123+, it is likely that CD123Bi armed ATC will be able to target CD33 positive leukemia stem cells. This approach may provide a non-toxic strategy to target leukemic blasts and leukemic stem cells. DisclosuresNo relevant conflicts of interest to declare.

Targeting advanced pancreatic cancer with activated t cells armed with anti-CD3 x anti-EGFR bispecific antibody.

4108Background: Conventional chemotherapy (CT) for advanced pancreatic cancer (PC) is associated with dismal response rates and poor survival. Arming anti-CD3 activated T cells (ATC) with anti-CD3 ...

Trial of anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) in patients with EGFR-expressing recurrent/metastatic non-small cell lung cancer (NSCLC) and solid tumors.

e13124 Background: Patients (pts) with advanced NSCLC and solid tumors have a poor prognosis and novel approaches to improve targeting and cytotoxicity directed at these cancers are needed. Anti-CD3 activated T cells (ATC) can be expanded and armed with anti-CD3 x anti-EGFR bispecific antibody (EGFRBi). Methods: In a phase I trial using ATC armed with EGFRBi, 6 pts (NSCLC = 5 pts, pancreatic cancer =1 pt) were treated with 8 infusions (twice/week) for 4 weeks with 5, 10 billion armed ATC/dose to determine the maximum tolerated dose (3 pts each). T cells from leukopheresis were activated and expanded with anti-CD3/IL-2 for 14 days, harvested, armed with EGFRBi and cryopreserved. IL-2 (3 x 105 IU/m2/daily) and GM-CSF (250 µg/m2 twice/week) was given beginning 3 days prior to the 1st infusion and ending++ 3, performance status (PS 0= 2 pts, PS 1= 4 pts). Results: Restaging scans revealed stable disease in 2 pts and progression in 4 pts. One pt with NSCLC at each dose level had stable disease (23 mths, 11 mths). ATC infusions were well tolerated and noticeable toxicities included chills (grade 2= 1 pt, grade 3= 1 pt); headache (grade 1= 2 pts, grade 2= 2 pts), dizziness (grade 2= 1 pt), nausea (grade 1= 2 pts). Accrual continues for dose levels at 20, 40 billion armed ATC per dose. No major toxicities with IL-2 were observed; bone pain resulted in dose reductions for GM-CSF in 2 pts. Conclusions: Infusions of EGFRBi armed ATC can be safely accomplished in patients with advanced NSCLC and solid tumors; accrual continues.

Targeting Cytomegalovirus-Infected Cells Using T Cells Armed with Anti-CD3 × Anti-CMV Bispecific Antibody

Human cytomegalovirus (CMV) reactivation and infection can lead to poor outcomes after allogeneic stem cell transplantation. We hypothesized that anti-CD3 activated T cells (ATCs) armed with chemically heteroconjugated anti-CD3 × polyclonal anti-CMV bispecific antibody (CMVBi) will target and eliminate CMV-infected cells. Arming doses of CMVBi as low as 0.01 ng/10(6) ATCs was able to mediate specific cytotoxicity (SC) directed at CMV-infected target cells significant above unarmed ATCs at mutiplicities of infection (MOI) between 0.01 and 1. At effector-to-target ratios (E:T) of 25:1, 12.5:1, 6.25:1, and 3.125:1, armed ATCs significantly enhanced killing of CMV-infected targets compared with unarmed ATCs. At an MOI of 1.0, the mean % SC directed at CMV-infected targets cells for CMVBi-armed ATCs at E:T of 3.12, 6.25, and 12.5 were 79%, 81%, and 82%, respectively; whereas the mean % SC for unarmed ATCs at the same E:T were all <20%. ATCs, Cytogam(®), or CMVBi alone did not lyse uninfected or CMV-infected targets. Co-cultures of CMVBi-armed ATCs with CMV-infected targets induced cytokine and chemokine release fromarmed ATCs. This nonmajor histocompatibility complex restricted strategy for targeting CMV could be used to prevent or treat CMV infections after allogeneic stem cell transplantation or organ transplantation.

Transfer of Cellular and Humoral Anti-Tumor Immunity with Immune T Cells After Stem Cell Transplant (SCT) for Metastatic Breast Cancer Following “Vaccination” with Anti-CD3 x Anti-Her2/Neu Bispecific Antibody (Her2Bi) Armed Activated T Cells

Abstract 1914Novel therapeutic approaches are needed for women with metastatic breast cancer (BrCa). In our phase I clinical trial, infusions of anti-CD3 activated T cells (ATC) armed with anti-CD3 x anti-Her2/neu bispecific antibody (Her2Bi) induced specific cytotoxicity (SC) directed at SK-BR-3 breast cancer cells by fresh peripheral blood lymphocytes (PBL) and induced elevated serum levels of Th1 cytokines. In this study, we took advantage of armed ATC induced anti-tumor immune responses by infusing “immune” T cells collected by leukopheresis. We expanded “immune T cells” with anti-CD3 and IL-2, followed by a cryopreservation for multiple re-infusions of ATC after high dose chemotherapy (HDC) and autologous stem cell transplant (SCT). We asked whether this approach would transfer anti-tumor responses back into patients after HDC and SCT to accelerate the development of cellular and humoral anti-tumor responses. This strategy of “prime” with armed ATC and “boost” with ATC was performed in 5 evaluable patients. The “priming” portion involved 8 infusions of (2 infusions/week for 4 weeks) armed ATC given with daily low dose IL-2 (300,000 IU/m2/day) and twice weekly GM-CSF (250 μg/m2). Approximately 3 weeks after the armed ATC infusions, patients underwent a second leukopheresis for the collection and expansion of ATC. The expanded ATC from 6 patients at an effector:target ratio (E/T) of 25:1 exhibited specific cytotoxicity (SC) ranging from 3.7–25.8 (mean = 13.6%) directed at the SK-BR-3. Phenotyping of the ATC showed a mean of 50.2 % (25–74) CD4+ cells, 30.4 % (16.3-51.3) CD8+ cells, 11.6% (4.5-24.3) CD56+CD16+ cells, and 29.5% (10.4–41) CD4+CD25+ cells. A separate leukopheresis after G-CSF stimulation was performed to obtain CD34+ cells for the SCT. After HDC and PBSCT, 5 evaluable patients received multiple infusions with a mean total of 54×109 ATC (16–110 × 109) beginning day +1 after SCT. No G-CSF was given to accelerate engraftment. There were no dose-limiting side effects or delays in engraftment. One patient developed sepsis, multiple organ failure and recovered fully with supportive care and antibiotics. Phenotyping at 2 weeks after SCT showed the mean proportions of CD4+ and CD8+ cells to be 55.5 and 17.7%, respectively. Specific cytotoxicity (SC) directed at SK-BR-3 targets ranged from 4.7 to 70% at E/T of 25:1 up to 18 months post SCT but not against a negative control cell line-Daudi. Mean serum anti-SK-BR-3 antibody levels were 800 ng/ml preSCT and 1500, 1080 and 1360 ng/ml at 1, 2, and 3 months post SCT, respectively. In vitro anti-SK-BR-3 antibody synthesis was assessed using a new assay (Thakur et al, Cancer Immunol Immunother EPub, 2011) showed easily detectable levels of in vitro anti-SK-BR-3 antibody synthesis. The mean anti-SK-BR-3 antibody synthesis in the presence of CpG in pre-immunotherapy (Pre-IT), mid-IT, 1 months post-IT, pre-SCT and 1, 2, and 3 months post-SCT is summarized in the table. PBL produced anti-SK-BR-3 antibody pre-SCT and there was clear recovery of anti-SK-BR-3 antibody synthesis by PBL at 2 and 3 months after SCT. These data show infusions of immune ATC transferred cytotoxic T lymphocytes and humoral antibody activity directed at tumor antigens. These novel findings suggest that adaptive immunity was transferred into patients by ATC infusions and the stem cell product after myeloablation and SCT leading to rapid reconstitution of anti-tumor immunity.Time PointsCulture ConditionsMean±SD (n=3)Pre-ITCpG23±27Mid-ITCpG66±671M Post-ITCpG41±67Pre-SCTCpG92±101M Post-SCTCpG9±182M Post-SCTCpG45±273M Post-SCTCpG82±55 Disclosures:Lum:Transtarget Inc: Equity Ownership, Founder of Transtarget.

Activated T cells from umbilical cord blood armed with anti‐CD3 × anti‐CD20 bispecific antibody mediate specific cytotoxicity against CD20+ targets with minimal allogeneic reactivity: a strategy for providing antitumor effects after cord blood transplants

In this study, we asked whether anti-CD3-activated T cells (ATCs) from cord blood (CB) could be expanded and targeted to solid tumors or hematologic malignancies for infusions after unrelated CB stem cell transplant and whether cord blood ATCs (CBATCs) could reduce alloresponsiveness. CB mononuclear cells (MNCs) were activated with anti-CD3 (20 ng/mL) and expanded for 14 days in interleukin-2 (100 IU/mL). CBATCs were armed with anti-CD3 × anti-CD20 (CD20Bi) or anti-CD3 × anti-Her2 (Her2Bi) bispecific antibodies (CBaATCs) and tested for specific cytotoxicity, cytokine secretion, and alloresponsiveness. Our results show the mean expansion of CBATCs to be 37-fold after 14 days of culture from either frozen (n=4) or fresh (n=4) CB units. Cytotoxicity was optimal when CBATCs were armed with 50 ng of CD20Bi/10(6) cells. Cytotoxicity peaked between Day 8 and Day 10 for both bispecific antibodies. At an effector-to-target ratio of 25:1, the mean cytotoxicities of CBATCs armed with Her2Bi or CD20Bi were 40% (n=4) and 30% (n=4), respectively. CBaATCs exhibited peak specific interferon-γ enzyme-linked immunosorbent spots on Day 10. CBATCs and CBaATCs suppressed responsiveness to alloantigens by 20% to 50% when compared with normal allogeneic peripheral blood MNC response. We showed that armed CBATCs mediate specific cytotoxicity, secrete low levels of cytokines and chemokines, and demonstrate attenuated response to alloantigens.

T-cells targeted with anti-CD3 x anti-HER2 bispecific antibody for treatment of women with stage IV breast cancer (phase I): Clinical and immune function results.

2548 Background: Nontoxic immunologic targeting strategies for the treatment of metastatic breast cancer (MBC) are needed. Anti-CD3 activated T cells (ATC) retargeted (armed) with anti-CD3 x anti-Her2 bispecific antibody HER2Bi) exhibit high levels of specific cytotoxicity, proliferate, and secrete cytokines/chemokines. This study was designed to test the safety, survival, persistence, and trafficking of aATC. Methods: We report a phase I trial in 23 patients (pts) with MBC (Her2 0-3+) who received escalating doses of 5, 10, 20, and 40 x 109Her2Bi armed ATC (aATC) twice per week for 4 weeks totaling 40, 80, 160, and 320 x 109 aATC with 300,000 IU/m2/day of IL-2 and GM-CSF 300 µg/m2 twice weekly beginning 3 days before the 1st infusion and ending 1 week after the last infusion. All 7 of the Her2 3+ pts had prior treatment with trastuzumab and most pts had multiple rounds of chemotherapy. Results: Grade III-IV toxicities (NC Immunotherapy Toxicity) by total episodes for 23 pts in decreasing order were chills (60), headache (17), nausea/vomiting (4), backpain (1) hypertension (1), and death (1) due to digoxin toxicity(1). Serum CEA in 3 of 5, CA27-29 in 4 of 4, and Her2 receptors in 4 of 5 pts tested decreased 15-50%. There was 1 partial response 7 months after therapy. Eleven had stable disease and 10 had progressive disease 12-14 weeks after starting aATC in 22 evaluable pts. The median overall survival was 34.5 months for the whole group, 68.8 months for the Her2 3+ group (7 pts), and 25.6 months for the Her 0-2+ group (15 pts). Infusions induced cytotoxicity directed at SK-BR-3 that persisted >4 months, a Th1 cytokine pattern, and increased IL-12p70 levels. aATC circulate for > 96 hrs and biopsies show that aATC localize to tumors. Conclusions: aATC infusions: 1) are safe; 2) induce endogenous cytotoxicity, anti-tumor antibody, and cytokine responses; 3) circulate for >96 hrs; and 4) traffic to tumors. These results suggest that aATC infusions may improve OS by breaking tolerance and “vaccinating” the pts against breast cancer antigens.

Targeting Lymphoma with Bispecific Antibody-Armed T Cells and their Potential for use in an Allogeneic Setting (41.31)

Abstract Patients with high-risk refractory or relapsed lymphomas face a poor prognosis and limited therapeutic options. Current treatment regimens of chemotherapy with either rituximab (anti-CD20 mAb) or with allogeneic stem cell transplant (alloSCT) yield serious toxicities and high relapse rates. In particular, benefits of alloSCT are grossly limited by life-threatening graft vs host disease (GvHD). New approaches to enhance graft vs lymphoma (GvL) effects without increased GvHD are needed. Bispecific antibodies (BiAbs) are produced by linking anti-CD3 for T cell binding to an antibody specific for a tumor antigen. Anti-CD3 activated T cells (ATC) "armed" with BiAb express tumor-specific CTL function independent of further activation, proliferation, antigen presentation or costimulation. Studies here examined allo-reactivity of BiAb-armed ATC for potential application in an allogeneic setting. Data show that ATC armed with anti-CD3 x anti-CD20 BiAb specifically target and kill B cell lymphomas. However, BiAb-armed ATC are remarkably poor stimulators of, or responders to, allo-stimlulation. These results provide important evidence of a functional separation between the GvL and GvH effects of BiAb-armed ATC.

Phase I immunotherapy in women with metastatic breast cancer with activated T cells targeted with anti-CD3 x anti-Her2/neu bispecific antibody

3034 Background: Women with metastatic breast cancer (MBC) have limited treatment options. Nontoxic approaches to improve targeting and cytotoxicity directed at MBC are needed. Anti-CD3 activated T cells (ATC) can be expanded and retargeted (armed) with anti-CD3 x anti-Her2 bispecific antibody (HER2Bi). Preclinical studies showed that Her2Bi-armed ATC exhibit high levels of specific cytotoxicity, proliferate, and secrete cytokines/chemokines (Clin Canc Res 12:569, 2006). Methods: In a phase I trial using ATC armed with Her2Bi, 17 MBC (31–86 yrs) patients (pts) were treated with 8 infusions (twice/week) for 4 weeks with 5, 10, 20, and 40 billion armed ATC/dose to determine the maximum tolerated or technically feasible dose. T cells from leukopheresis were activated and expanded with anti-CD3/IL-2 for 14 days, harvested, armed with Her2Bi at 50 ng/106ATC, and cryopreserved. IL-2 (3 x 105 IU/m2/daily) and GM-CSF (250 μg/m2 twice/week) was given beginning 3 days prior to the 1st infusion and ending 1 week after the last infusion. Results: The toxicities are summarized in the Table. Restaging at 1 month revealed 1 pt who developed a PR, 9 pts with SD, 5 pts with PD. Two pts were not evaluable. Three of 5 with CA27–29 markers decreased their markers by 15–50%. In 4 patients with positive CEA markers, 2 reduced their CEA levels by >50% and 2 by >15–50%. In 5 pts with circulating Her2 receptors (Her2r), 2 decreased Her2r by >50% and 2 reduced their Her2 by >15–50%. The median follow-up was 14.9 mos for the HER2(3+) group and 15.7 mos for the HER2(0–2+) group. Median time to progression was 3 and 6 mos for the HER2(3+) and HER2 (0–2+) groups, respectively. In the HER2(3+) group, median overall survival was not been reached with median follow-up of 14.9 mos. The median survival for the HER2(0–2+) cohort is 21.3 mos. The median survival for all pts remains undefined. Conclusions: These data suggest that infusions of armed targeted T cells can be safely infused and may improve overall survival in women with MBC. Infusion Toxicities Grade I II III IV # Episodes Chills 0 4 36 0 40 Headache 0 3 14 1 17 Nausea/Vomiting 8 1 0 0 9 Fever 3 1 0 0 4 Hypotension 1 3 0 0 4 Hypertension * 0 0 0 1 1 Dyspnea 0 1 0 0 1 # Episodes/130 infusions; * hypertension associated with a subdural hematoma. 1 pt died of Digoxin toxicity Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Transtarget Transtarget Transtarget

Induction of Immune Responses and Improved Survival after Infusions of T Cells Armed with Anti-CD3 X Anti-Her2/Neu Bispecific Antibody in Stage IV Breast Cancer Patients (Phase I).

Women with stage IV metastatic breast cancer (mBrCa) have limited treatment options since toxicities from chemotherapy and radiotherapy become limiting. Non-toxic immunotherapy approaches to improve targeting and cytotoxicity directed at breast cancer are needed. Our earlier study showed that anti-CD3 activated T cells (ATC) could be expanded in culture and then armed with anti-CD3 × anti-Her2/neu bispecific antibody (HER2Bi). The armed ATC mediate enhanced specific cytotoxicity, proliferate, and induce cytokine/chemokine secretion (J Hematotherapy & Stem Cell Res 10:247, 2001). In a Phase I trial using ATC armed with Her2Bi, 18 Stage IV BrCa patients (pts) were treated with 8 infusions (twice/week) for 4 weeks totaling 40 (6 pts), 80 (2 pts), 160 (7 pts), and 320(1 pt) billion ATC armed with Her2Bi without dose-limiting toxicities. The most frequent side-effects were chills, fever, and hypotension that were easily controlled with medications. Two stage IV mBrCa patients had minor responses with decreases in CEA (35.2 to 4.1 ng/ml) or CA 27-29 (57.7 to 35.6 U/ml) and one pt had partial response with a decreased liver metastatic lesion. None of the pts developed human anti-mouse antibodies levels above10 ng/ml. Immunoaffinity-depletion of BiAb-armed ATC from PBMC of a high risk IV mBrCa pt at post-treatment time points showed an increase in anti-BrCa tumor cell activity exhibited by endogenous immune cells that persisted up to 4 months after treatment. Increasing proportions and absolute numbers of CD25+ cells in CD4+ and CD8+ subsets were observed as a function of treatment with nearly all CD4+ and CD8+ cells being CD25+ by 1 week post-final infusion. Significant treatment-associated elevations (several log increases over baseline) of circulating IFNγ, TNFα, IL-2, IL-5, IL-10, IL-12p70, and IL-13 were detected in serum of nearly all of the patients beginning 1–2 weeks after initiation of infusions. Particularly remarkable was the 3 log increase of mean (n=9) serum IL-12p70 from 0 to 1200 pg/ml. There was a Th1 shift that persisted during therapy. To date, results from the phase I clinical trial suggest that Her2Bi-armed ATC activate the endogeneous immune system to generate an adaptive immune responses despite the presence of high tumor burdens. The figure shows the overall survival for 18 women (All) treated on the phase I protocol with the median survival not yet defined for the HER2/neu 3+ group and the entire study group. The median survival for the 9 pts with Her2/neu negative disease was 21.5 months. Together these data are encouraging and strongly suggest infusions of armed targeted T cells may immunized the patient against their own tumor antigens leading to immunoreactivity manifested as the development of a persistent CTL response that may lead to improved overall survival. [Display omitted]

Anti-CD3 Activated T Cells from Cord Blood Can Be Expanded Ex Vivo and Armed with Bispecific Antibodies To Lyse Her2/Neu + and CD20+ Targets: A Strategy for Providing Anti-Tumor/Lymphoma Effects after Cord Blood Transplant.

Our previous studies showed that anti-CD3 activated T cells (ATC) from peripheral blood mononuclear cells could be expanded in interleukin-2 (IL-2) for 14 days and armed with anti-CD3 x anti-Her2/neu (Her2Bi)[J Hemat & Stem Cell Res 10:247, 2001], anti-CD3 x anti-CD20 (CD20Bi)[Exp Hemat 33:452, 2005], or anti-CD3 x anti-EGFR [Clin Cancer Res 12:183, 2006] bispecific antibody (BiAb)and can kill Her2/neu, CD20, and EGFR+ tumor targets, respectively. In this study, we asked whether anti-CD3 activated cord blood T cells (CBATC) could be expanded and targeted with Her2Bi and CD20Bi to tumors or hematologic malignancies for infusions after cord blood stem cell transplant (CBSCT). CB mononuclear cells were activated with anti-CD3 (20 ng/ml) and expanded for 14 days in IL-2 (100 IU/ml). CBATC were armed with Her2Bi or CD20Bi and tested for specific cytotoxicity directed SK-BR-3, Raji, or B9C targets and cytokine secretion or IFNγ EliSpots after binding to tumor cells. Our results show the mean expansion of CBATC to be 43-fold (n=8) after 14 days of culture. By the end of culture, the proportions of CD8+ and CD4+ were 82% and 18%, respectively. The proportion of cells expressing CD19 or CD20 did not exceed 6.3%, CD56+ cells were <3.6% and CD3-CD16+CD56+ cells was <0.7%. Cells positive for CD4+CD25+ or CD8+ CD25+ were 4.2% or 7.1%, respectively (n=2). Specific cytotoxicity was optimized when CBATC were armed with 50 ng/106 cells of Her2Bi or CD20Bi (arming dose ranged from 5, 50, and 500 ng/106 cells); arming significantly increased cytotoxicity of the armed CBATC over that seen for unarmed CBATC. Cytotoxicity peaked between days 12 and 14 for both BiAbs. The ability of CD20Bi armed ATC to produce Elispots for IFNγ were tested by arming ATC the CD20Bi after 0, 8, 11, and 13 days of culture peaked on day 8. Only CD20+ targets induced Elispots and day 8 armed ATC exhibited peak numbers (2,200 Elispots(ranged from 1,700 to 1,800 on day 8)/106 armed ATC plated). At an effector/target ratio (E:T) of 25:1, the mean cytotoxicity of CBATC armed with Her2Bi or CD20Bi was 60% (n=4) and 35% (n=1), respectively. In an extended culture to day 47, mean cytotoxicity for Her2Bi-armed CBATC was 36% at an E/T of 25:1 compared to 4.35% for unarmed CBATC. Unarmed CBATC did not kill Daudi targets. Armed CBATC mediated both specific cytotoxicity and secreted IFN-γ as measured by ELISA or EliSpots. Both fresh and frozen CB could be used in the assays. In a clinical application, specific cytotoxicity of armed CBATC could be used to augment anti-tumor and anti-lymphoma effects after CBSCT.

Anti-CD3 × Anti-Epidermal Growth Factor Receptor (EGFR) Bispecific Antibody Redirects T-Cell Cytolytic Activity to EGFR-Positive Cancers In vitro and in an Animal Model

Targeting epidermal growth factor receptor (EGFR) overexpressed by many epithelial-derived cancer cells with anti-EGFR monoclonal antibodies (mAb) inhibits their growth. A limited number of clinical responses in patients treated with the anti-EGFR mAb, (cetuximab), may reflect variability in EGFR type or signaling in neoplastic cells. This study combines EGFR-targeting with the non-MHC-restricted cytotoxicity of anti-CD3 activated T cells (ATC) to enhance receptor-directed cytotoxicity. ATC from normal and patient donors were expanded ex vivo. Specific cytolytic activity of ATC armed with anti-CD3 x anti-EGFR (EGFRBi) against EGFR-expressing cancer cells derived from lung, pancreas, colon, prostate, brain, skin, or EGFR-negative breast cancer cells was evaluated in (51)Cr release assays. In vivo studies comparing tumor growth delay induced by EGFRBi-armed ATCs or cetuximab were done in severe combined immunodeficient/Beige mice (SCID-Beige) bearing COLO 356/FG pancreatic and LS174T colorectal tumors. At effector/target ratios from 3.125 to 50, both EGFRBi-armed normal and patient ATC were significantly more cytotoxic, by 23% to 79%, against EGFR-positive cells over ATC, cetuximab, anti-CD3 alone, or ATC armed with irrelevant BiAb directed at CD20. EGFRBi-armed ATC also secreted significantly higher levels of some T(H1)/T(H2) cytokines compared with ATC alone. In mice, i.v. infusions of EGFRBi-armed ATC (0.001 mg equivalent/infusion) were equally effective as cetuximab (1 mg/infusion) alone for significantly delaying growth of established COLO 356/FG but not LS174T tumors compared with mice that received ATC alone or vehicle (P < 0.001). Combining EGFR antibody targeting with T cell-mediated cytotoxicity may overcome some limitations associated with EGFR-targeting when using cetuximab alone.

Infusions of T Cells Armed with Anti-CD3 x Anti-Her2/neu Bispecific Antibody Modulate In Vivo Patient Immune Responses in Phase I Clinical Trials for Breast and Hormone Refractory Prostate Cancers.

Phase I dose escalation trials using ex vivo expanded, anti-CD3 activated T cells (ATC) armed with anti-CD3 x anti-Her2/neu (Her2Bi) to evaluate toxicity and immune responses were conducted in 8 Stage IV breast cancer (BrCa), 5 high-risk Stage II BrCa, and 3 hormone refractory prostate cancer (HRPC) patients. Patients received 8 infusions (Inf) twice per week for 4 weeks totaling 40 billion (11/16 patients) or 80 billion (5/16 patients) HER2Bi-armed ATC. Phenotyping the expansion product showed 90.0±10.1% (mean±SD)CD3, 47.5±22.2 CD4, and 41.9±19.9 CD8. Specific lysis of SK-BR-3 targets by patients' armed ATC (n= 16 patients) at E:T of 10, 5, and 2.5 averaged 54.1±17, 39.5±14, and 25.9±11, respectively. These results were comparable to 63.4±17 (p = 0.23), 57.7±20 (p = 0.09), and 33.9±16 (p = 0.38) specific lysis mediated by armed ATC from 6 normal donors at the respective E:T confirming the function of Her2Bi-armed ATC at the time of Inf. By ELISA, 4/8 (50%) of the patients developed anti-mouse antibody responses (HAMA) to OKT3, which peaked (median: 35.4 ng/ml; range: 4–64 ng/ml) between Inf#5 and Inf#8 (14–27 days post-Inf #1). There were no AEs associated with elevated HAMA titers. Th1/Th2 panels were obtained from patient sera using the Bio-Plex Protein Array System and analyzed as a function of number of Infs. The in vivo T cell response, calculated as the mean Th1[IL-2+IFN-g]/Th2[IL-4+IL-5] ratio of cytokines, showed a polarized Th1 response as a function of armed ATC Inf that increased from 89.1(Inf#1) to 538.6 (Inf #8). Levels of IL-12 also increased by Inf#5 and were constant from Inf#5 through Inf#8 (mean±SD = 34.0±17.2 pg/ml). These findings were consistent with increased CTL responses observed in ELISPOTs of patient post-Inf PBMC exposed to Her2+ tumor targets. Overall immune response, calculated as the average ratio of Th1[IL-2+IFN-g] /Th2 [IL-4+IL-5+IL-10+IL-13], remained Th1 polarized throughout treatment though the ratio decreased as a function of Inf#: going from 9.46 (Inf#1) to 1.42 (Inf #8). TNF-alpha levels were contant from Inf #1–8 (mean±SD = 9.5 ± 4.9 pg/ml), and GM-CSF peaked between Inf #5–8 (92.2 ± 36.4 pg/ml). Detection of MIgG2a+ (anti-CD3 coated) cells in the PBL from 8 patients was highest post-Inf#1 (18.4% ± 16.1) and decreased by Inf #5–8 (4.1% ± 6.1). In 2 patients, MIgG2a+ armed T cells could still be detected 1 week and 3 weeks after Inf#8. All patients received their targeted doses of armed ATC: no dose-limiting toxicities were observed in any patients, and with the exception of a few patients with grade II–III chills/grade II fever, infusions of Her2Bi-armed ATC were well-tolerated. These observations provide evidence that the endogenous immune system is being modulated by the infusion of armed ATC and further support the rationale for performing phase II trials using armed ATC in Her2+ advanced/high risk BrCa and HRPC patients.

Targeting of Lin−Sca+ hematopoietic stem cells with bispecific antibodies to injured myocardium

Bispecific antibodies (BiAbs) are being used to target T cells or other immune cells to antigen-specific tumor targets. Anti-CD3 activated T cells (ATC) armed with anti-CD3 × anti-HER2 BiAb (HER2Bi) have been used to target Her2/ neu + breast and prostate carcinoma cells. We adapted BiAb technology to target stem cells to injured myocardium. Since myocardial infarctions can lead to cardiac death and disability, rapid repair and rejuvenation of damaged myocardium is critically needed. Effective homing of stem cells and transdifferentiation of the stem cells into functional elements of the myocardium is needed for repair of damaged myocardium. We use a BiAb that binds c-kit on murine stem cells and VCAM-1 adhesion molecules up-regulated on injured myocardial cells. To test for specific binding and homing in a mouse, we produced anti-c-kit × anti-VCAM-1 to target purified Lin−Sca+ murine stem cells to the injured myocardium. Mice with infarcts created by ligation of the left anterior descending artery (LAD) were directly injected with armed stem cells or injected via the internal jugular vein (IJ) with FACS sorted Lin−Sca+ stem cells from bone marrow after fluorescent dye labeling. There were increased numbers of armed Lin−Sca+ cells retained in infracted myocardium after direct injection of armed Lin−Sca+ cells and increased numbers of Lin−Sca+ cells that were found in injured myocardium after IJ injection. These results suggest that stem cells retargeted with BiAb can be directly injected and retained by injured myocardium or targeted to injured myocardial tissues for tissue regeneration.