Tissue Cross-reactivity Research Articles

Abstract 2802: Fine-tuning T cell receptors for adoptive T cell therapy

Abstract Adoptive T cell therapy (ACT) with gene-modified T cells is emerging as a highly promising strategy for the treatment of many types of cancer. Therapies associated with the induction of anti-tumor immunity, such as the administration of vaccines or the ex-vivo expansion of patient-derived TILs, have historically had limited success. This lack of efficacy is almost certainly brought about as a consequence of thymic selection, leading to a paucity of T cells, and T cell receptors, in the circulating repertoire, with a sufficient degree of sensitivity towards tumor-associated antigens. An alternative approach, which has generated promising results, relies on the adoptive transfer of patient-derived T cells that have been first modified to express a chimeric antigen receptor (CAR). Nevertheless, a lack of tumor-specific antibody targets limits the wider applicability of CAR T cells. To overcome these issues we have developed a strategy that involves the transfer of T cells expressing engineered TCRs with optimised affinity for a defined tumor antigen. Since TCRs recognize peptide antigens presented on the cell surface in complex with human leukocyte antigen (HLA), they give access to the large pool of intracellular proteins. Furthermore, the use of TCRs means that the natural interaction between a T cell and the corresponding antigen presenting cell is preserved. Importantly, for any given antigen, engineered TCRs require individual optimization, to ensure the appropriate balance between potency and specificity. Recent and emerging clinical data using engineered TCRs for ACT are beginning to corroborate promising pre-clinical findings, with evidence of both potent and durable responses in vivo. However, experiences have also highlighted the dangers of this approach. The main risks associated with TCR adoptive therapy are target presentation on healthy tissue and TCR cross-reactivity to a look-alike peptide. Understanding these risks and ensuring comprehensive and robust safety testing mechanisms are in place is pivotal to mitigate these risks. Citation Format: Bent Jakobsen. Fine-tuning T cell receptors for adoptive T cell therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2802. doi:10.1158/1538-7445.AM2014-2802

Can we unlock the potential of IGF-1R inhibition in cancer therapy?

IGF-1R inhibitors arrived in the clinic accompanied by optimism based on preclinical activity of IGF-1R targeting, and recognition that low IGF bioactivity protects from cancer. This was tempered by concerns about toxicity to normal tissue IGF-1R and cross-reactivity with insulin receptor (InsR). In fact, toxicity is not a show-stopper; the key issue is efficacy. While IGF-1R inhibition induces responses as monotherapy in sarcomas and with chemotherapy or targeted agents in common cancers, negative Phase 2/3 trials in unselected patients prompted the cessation of several Pharma programs. Here, we review completed and on-going trials of IGF-1R antibodies, kinase inhibitors and ligand antibodies. We assess candidate biomarkers for patient selection, highlighting the potential predictive value of circulating IGFs/IGFBPs, the need for standardized assays for IGF-1R, and preclinical evidence that variant InsRs mediate resistance to IGF-1R antibodies. We review hypothesis-led and unbiased approaches to evaluate IGF-1R inhibitors with other agents, and stress the need to consider sequencing with chemotherapy. The last few years were a tough time for IGF-1R therapeutics, but also brought progress in understanding IGF biology. Even failed studies include patients who derived benefit; they should be investigated to identify features distinguishing the tumors and host environment of responders from non-responders. We emphasize the importance of incorporating biospecimen collection into trial design, and wording patient consents to allow post hoc analysis of trial material as new data become available. Such information represents the key to unlocking the potential of this approach, to inform the next generation of trials of IGF signalling inhibitors.

A CD19/CD3 Bispecific Tandab, AFM11, Recruits T Cells To Potently and Safely Kill CD19+ Tumor Cells

To harness the potent tumor-killing capacity of T cells for the treatment of CD19+ malignancies, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 and CD19, the CD19/CD3 RECRUIT-TandAb AFM11. CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma. Since native antibodies cannot recruit T cells, we engineered a bispecific anti-CD19/anti-CD3 TandAb. The tumor-specific CD19 antigen module targets the TandAb to cancer cells, while simultaneously, the CD3 effector module recruits and activates T cells, leading to cancer cell lysis. The advantages of the TandAb technology, relative to other bi-functional fragment antibody scaffolds, include: improved pharmacokinetics (PK) enabling intravenous dosing, more drug-like properties, and avidity-enhanced efficacy for the targeting and killing of tumor cells. We evaluated in vitro efficacy and safety using CD19+ cell lines, and in vivo efficacy in a murine NOD/scid xenograft model reconstituted with human PBMC. Further, we used standard preclinical IND enabling assays to evaluate tissue cross reactivity, PK, and toxicological profile (local tolerance, hematocompatibility, effects on hematopoesis, etc).In vitro assays demonstrated the higher potency and efficacy of target cell lysis by AFM11 relative to a bispecific tandem scFv (that is currently in clinical evaluation). CD8+ T cells dominate early AFM11-mediated cytotoxicity (4 hrs) while after 24 hrs both CD4+ and CD8+ T cells equally contribute to tumor lysis with EC50 between 0.5 – 5 pM; cytotoxicity was independent of CD19 cell-surface density. AFM11 exhibited similar cytotoxicity over effector:target ratios ranging from 5:1 to 1:5, and facilitated serial T cell-killing of its targets. The advantage of AFM11 over the bispecific tandem scFv was most pronounced at lower effector:target ratios. AFM11 activated T cells only in the presence of CD19+ cells. In PBMC cultures, AFM11 induced CD69 and CD25 expression, T cell proliferation, and production of IFN-γ, TNF-α, IL-2, IL-6, and IL-10. Depletion of CD19+ cells from PBMC abrogated these effects, demonstrating that the T cell activation is strictly CD19+ target-dependent. Thus, AFM11 should not elicit the devastating cytokine release observed when full-length antibodies bind CD3. Up to one week co-incubation with AFM11 did not inhibit T cell cytotoxicity, suggesting that the TandAb does not induce anergy. In vivo, AFM11 induced dose-dependent growth inhibition of Raji tumors; a single 0.5 mg/kg dose exhibited efficacy similar to 5 daily injections. In the tissue cross reactivity study, only tissues containing CD19+ and CD3+ cells were stained by AFM11; all other tissues, including vital organs, displayed no cross reactivity. Similarly, no local intolerance was observed in rabbits, and no effect on myeloid and erythroid progenitors was observed in a colony-forming assay. Strong accumulation of 125I-labeled AFM11 was observed in the tumors of mice engrafted with CD19+ cancer cells, and no unspecific organ accumulation was observed. Finally, evaluated on the basis of Cmax and the area under the curve (AUC), AFM11 exhibited dose linearity (20 – 500 mg AFM11 dose range) upon single i.v. bolus administration in mice; half-life (T1/2) ranged from 18.4 to 22.9 hr.In summary, AFM11 is a highly efficacious novel drug candidate for the treatment of CD19+ malignancies with an advantageous safety profile and anticipated dosing regimen. Disclosures:Zhukovsky:Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment. Little:Affimed Therapeutics AG: Consultancy, Equity Ownership.

Considerations for the nonclinical safety evaluation of antibody drug conjugates for oncology

Antibody drug conjugates (ADCs) include monoclonal antibodies that are linked to cytotoxic small molecules. A number of these agents are currently being developed as anti-cancer agents designed to improve the therapeutic index of the cytotoxin (i.e., cytotoxic small molecule or cytotoxic agent) by specifically delivering it to tumor cells. This paper presents primary considerations for the nonclinical safety evaluation of ADCs and includes strategies for the evaluation of the entire ADC or the various individual components (i.e., antibody, linker or the cytotoxin). Considerations are presented on how to design a nonclinical safety assessment program to identify the on- and off-target toxicities to enable first-in-human (FIH) studies. Specific discussions are also included that provide details as to the need and how to conduct the studies for evaluating ADCs in genetic toxicology, tissue cross-reactivity, safety pharmacology, carcinogenicity, developmental and reproductive toxicology, biotransformation, toxicokinetic monitoring, bioanalytical assays, immunogenicity testing, test article stability and the selection of the FIH dose. Given the complexity of these molecules and our evolving understanding of their properties, there is no single all-encompassing nonclinical strategy. Instead, each ADC should be evaluated on a case-by-case scientifically-based approach that is consistent with ICH and animal research guidelines.

Regulatory Forum Opinion Piece*

In the context of monoclonal antibody drug development, tissue cross-reactivity (TCR) studies are immunohistochemical (IHC) studies conducted on a broad panel of normal body tissues, using the antibody therapeutic as the primary reagent. The ICH S6 guidance states that these studies are performed to support investigational new drug (IND) or clinical trial applications (CTAs) for the initial dosing of human subjects for all antibody-based therapeutics. The purpose of TCR studies is, ostensibly, to inform investigators and regulators of potential target organs of toxicity based upon the binding of the therapeutic agent to tissue components. The value of TCR studies in predicting toxicity that would not otherwise have been detected in in vivo pharmacology and toxicology studies in pharmacologically relevant animal models has been a subject of debate for more than a decade. The history, challenges, and proper and improper uses of TCR studies were reviewed in this forum by Leach et al. in 2010. Coincident with this publication, a cross-industry survey on the utility of TCR assays in predicting human toxicity was separately published by a working group of the preclinical safety committee of the Biotechnology Industry Organization

Engineering and Characterization of the Chimeric Antibody That Targets the C-terminal Telopeptide of the α2 Chain of Human Collagen I: A Next Step in the Quest to Reduce Localized Fibrosis

Inhibition of the extracellular process of collagen fibril formation represents a new approach to limiting posttraumatic or postsurgical localized fibrosis. It has been demonstrated that employing a monoclonal antibody that targets the C-terminal telopeptide of the α2 chain of collagen I blocks critical collagen I–collagen I interaction, thereby reducing the amount of collagen deposits in vitro and in animal models. Here, we developed a chimeric variant of a prototypic inhibitory antibody of mouse origin. The structure of this novel antibody was analyzed by biochemical and biophysical methods. Moreover, detailed biochemical and biological studies were employed to test its antigen-binding characteristics. The ability of the chimeric variant to block formation of collagen fibrils was tested in vitro and in high-density cultures representing fibrotic processes occurring in the skin, tendon, joint capsule, and gingiva. The potential toxicity of the novel chimeric antibody was analyzed through its impact on the viability and proliferation of various cells and by testing its tissue cross-reactivity in sets of arrays of human and mouse tissues. Results of the presented studies indicate that engineered antibody-based blocker of localized fibrosis is characterized by the following: (1) a correct IgG-like structure, (2) high affinity and high specificity for a defined epitope, (3) a great potential to limit the accumulation of collagen-rich deposits, and (4) a lack of cytotoxicity and nonspecific tissue reactivity. Together, the presented study shows the great potential of the novel chimeric antibody to limit localized fibrosis, thereby setting ground for critical preclinical tests in a relevant animal model.

Abstract 2851: Development of an alkalizing antibody-enzyme conjugate for NSCLC treatment that is in Phase I clinical testing.

Abstract Many solid human tumors generate an acidic and hypoxic microenvironment as a result of altered metabolic pathways and aberrant tumor vasculature. In certain tumors, the chronic exposure to acidic extracellular conditions has been reported to promote invasiveness and metastatic behaviour. In addition, the lower pH may promote resistance to weakly basic chemotherapeutic agents by altering their partitioning coefficient between the extracellular and intracellular compartments. L-DOS47, an immunoconjugate cancer therapeutic, has been developed to target this unique tumor microenvironment. L-DOS47 is a conjugate of a lung adenocarcinoma specific single domain antibody and a urease enzyme. The antibody serves as a targeting agent to deliver the enzyme to the affected site while the urease enzyme coverts urea, an abundant metabolite, into ammonia and generates a local pH increase. The combined effect of ammonia toxicity and pH increase is cytotoxic to cancer cells as shown both in culture and in xenograft models. In vitro experiments showed that L-DOS47 also dramatically potentiated the cytotoxicity of a number of chemotherapeutics. Imaging studies using A549 xenografts and labelled L-DOS47 applied intravenously showed that the drug molecule preferentially accumulated and persisted at the tumor site. L-DOS47 has been studied in a number of tissue-cross reactivity studies to identify suitable animal species for toxicological studies. Cryosections of normal human, cynomolgus monkey, Sprague-Dawley rat, and CD-1 mouse tissues were compared. L-DOS47 produced specific staining to control material (BxPC-3 cells) and those tissues that are known to express CEACAM6 - an antigen for L-DOS47. Both cytoplasmic and membrane bound staining were observed. Results of these studies suggest that the primate and both rodent species are suitable for animal toxicological studies. Pivotal GLP compliant animal toxicological studies in cynomologus monkey and rat together with several non-GLP pilot animal studies were done. In the primate GLP study, L-DOS47 (0, 17, 26, and 35 μg/kg) was administered by IV infusion on Days 1, 8, 15 and 22, followed by a 28-day recovery period. Main study animals were sacrificed on Day 25 and recovery animals on Day 50. Standard toxicology parameters were evaluated. In addition, toxicokinetics, cytokine stimulation and immunogenicity were also evaluated. A separate cardiovascular safety pharmacology study was also conducted. No treatment related clinical signs were observed for the animals treated with 17 or 26μg/kg of L-DOS47. Adverse signs were observed in some but not all of the high dose (35 μg/kg) treated animals. Based on adverse clinical signs observed at 35μg/kg, the NOAEL was determined to be 26 μg/kg/day in this study. Currently L-DOS47 is approved for phase I/II studies in Europe and the U.S.A. Patient enrollment has begun in Poland and first patient has been dosed. Citation Format: Heman Chao, Baomin Tian, Kim Gaspar, John Docherty, Wah Wong. Development of an alkalizing antibody-enzyme conjugate for NSCLC treatment that is in Phase I clinical testing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2851. doi:10.1158/1538-7445.AM2013-2851

A novel treatment for ovarian cancer (OC): Anti-Müllerian inhibiting substance type II receptor (MISRII) humanized monoclonal antibody (mAb) 3C23K—Preclinical validation.

5069 Background: Expressed on most OC subtypes while displaying a restricted expression profile in adult normal tissues, MISRII represents a potentialtarget for OC immunotherapy. We present here the preclinical assessment of a humanized anti-MISRII EMABling mAb, 3C23K. Methods: Either quantitative RT-PCR or immunohistochemistry (IHC) studies were performed to confirm MISRII expression profile in Granulosa Cell Tumor (GCT) or Epithelial OC (EOC) patient samples and to evaluate tissue cross-reactivity. For in vitro and in vivo experiments, we have generated 4 patient-derived MISRII expressing EOC cell lines. Xenograft studies were conducted in swiss nude mice on established tumors (100 mm3). Mice received 2 to 3 weekly i.p. injections (10 mg/kg/inj) for 4 to 6 wks and tumor volumes were compared with control groups. Comparison of i.p. vs i.v. injections were assessed as well as combination with carboplatin (once a week for 4 weeks, 60 mg/kg/inj). In addition, 3C23K plasma level was monitored to determine half-life. Results: 1) Target validation: we confirmed by IHC the expression of MISRII in most OC tissue sections (4/4 GCT and 13/14 EOC), meanwhile, MISRII mRNA was only detected in 7/48 normal tissues. 2) In vitro assessment: tested in vitro 3C23K displayed both cytotoxic (ADCC) and anti-proliferative activities. 3) In vivo assessment: in the mouse xenograft models 3C23K exhibited a strong anti-tumoral activity as measured by tumor volume, with T/C ratios reaching values below 0.42 shortly after the initiation of treatment. No differences in efficacy were noticed between i.p. and i.v. injections or between thrice vs twice a week administrations. In addition, similar half lives were observed for 3C23K injected either i.v. (96.9 h) or i.p (113.5 h). Finally, the combination of 3C23K with carboplatin (CP), a standard of care in OC, exhibited an even stronger anti-tumor activity with T/C values at D22 of 0.06 (3C23K+CP), 0.18 (3C23K) and 0.69 (CP) vs vehicle. Conclusions: 3C23K represents a promising candidate for OC targeted therapy and a dose-escalation phase I study is planned in patients with OC.

The Use of Specific Monoclonal Antibodies to Target Immunogenic Tumor Membrane Proteins in Patients with Recurrent Pancreatic and Colon Cancer

Tumor associated antigens from pooled allogeneic membrane proteins were isolated, partially purified and tested as a possible vaccine in patients with stage II and III colon cancer. The vaccine, when given in combination with an adjuvant following surgical resection, resulted in marked improvement in survival compared to control patients having only undergone surgical resection of their tumor. While it was possible to demonstrate that patients receiving vaccine turned on both humoral and cell mediated responses, it appears that survivors remaining free of disease at 5-7 yrs post op were able to mount a strong IgG1 response as the primary mechanism for tumor destruction. Antibodies from hybridomas made against the vaccines resulted in production of monoclonals with a high degree of ADCC. Those monoclonals targeting pancreatic cancer and in particular the MUC5ac mutated antigen representing tumor immunogen were studied in detail. Animal models indicated rapid tumor destruction when nude mice, injected with human pancreatic cancer were then immunized with NPC-1 monoclonal antibody targeting mutated MUC5ac. FDA studies including tissue cross reactivity, biodistribution, and cytokine release assays indicated safety and efficacy of the monoclonals we have developed. Submission of the IND allowed for initiation of the Phase I trial using mAb NPC-1 targeting pancreatic cancer when that antigen was found to be expressed.

Preclinical Pharmacokinetics and Safety of Sym004: A Synergistic Antibody Mixture Directed against Epidermal Growth Factor Receptor

Sym004 is a novel therapeutic antibody mixture product comprising two unmarketed monoclonal antibodies (mAb) targeting the epidermal growth factor receptor (EGFR). In previous preclinical proof-of-concept studies, Sym004 was shown to elicit superior cancer cell growth inhibition activities compared with marketed anti-EGFR mAbs. This article describes the design and results of the preclinical safety program conducted to support early clinical development of Sym004. Tissue cryosections from various species were stained with Sym004 to evaluate tissue cross reactivity. The pharmacokinetics of Sym004 were evaluated in a mouse xenograft model and in Cynomolgus monkeys. Monkeys received once weekly intravenous infusions of Sym004 in the range 2 to 24 mg/kg for 6 to 8 weeks. Cetuximab (a marketed anti-EGFR mAb) and the individual antibodies comprising Sym004 were included in the repeat-dose toxicity studies at single-dose level. Sym004 had a staining pattern similar to cetuximab in tissue panels from both human and non-human primates. Once weekly dosing of Sym004 to Cynomolgus monkeys did not cause accumulation, whereas administration of the individual antibodies resulted in prolonged half-life and accumulation. In direct comparisons with cetuximab, Sym004 did not induce any distinct or novel adverse findings in the animals. However, an early onset of pronounced, reversible, and anticipated anti-EGFR-mediated pharmacologic effects, such as skin rash, dehydration, and liquid feces, was observed. Only minor adverse effects were recorded in animals treated with the individual antibodies comprising Sym004. Sym004 was well tolerated and did not induce any unexpected toxicities. The preclinical safety data enabled initiation of the ongoing clinical development.

Humanized anti-Trop-2 IgG-SN-38 conjugate for effective treatment of diverse epithelial cancers: preclinical studies in human cancer xenograft models and monkeys.

Evaluate the efficacy of an SN-38-anti-Trop-2 antibody-drug conjugate (ADC) against several human solid tumor types, and to assess its tolerability in mice and monkeys, the latter with tissue cross-reactivity to hRS7 similar to humans. Two SN-38 derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to the anti-Trop-2-humanized antibody, hRS7. The immunoconjugates were characterized in vitro for stability, binding, and cytotoxicity. Efficacy was tested in five different human solid tumor-xenograft models that expressed Trop-2 antigen. Toxicity was assessed in mice and in Cynomolgus monkeys. The hRS7 conjugates of the two SN-38 derivatives were equivalent in drug substitution (∼ 6), cell binding (K(d) ∼ 1.2 nmol/L), cytotoxicity (IC(50) ∼ 2.2 nmol/L), and serum stability in vitro (t/(½) ∼ 20 hours). Exposure of cells to the ADC demonstrated signaling pathways leading to PARP cleavage, but differences versus free SN-38 in p53 and p21 upregulation were noted. Significant antitumor effects were produced by hRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P ≤ 0.05), Capan-1 (P < 0.018), BxPC-3 (P < 0.005), and COLO 205 tumors (P < 0.033) when compared to nontargeting control ADCs. Mice tolerated a dose of 2 × 12 mg/kg (SN-38 equivalents) with only short-lived elevations in ALT and AST liver enzyme levels. Cynomolgus monkeys infused with 2 × 0.96 mg/kg exhibited only transient decreases in blood counts, although, importantly, the values did not fall below normal ranges. The anti-Trop-2 hRS7-CL2A-SN-38 ADC provides significant and specific antitumor effects against a range of human solid tumor types. It is well tolerated in monkeys, with tissue Trop-2 expression similar to humans, at clinically relevant doses, and warrants clinical investigation.

Abstract 1752: Nonclinical safety and pharmacokinetics of Sym004: A synergistic anti-EGF receptor antibody mixture

Abstract Epidermal growth factor receptor (EGFR) is a well validated oncology target for monoclonal antibodies (mAb's). Symphogen is developing a novel and more efficacious anti-EGFR therapy to treat cancer patients with EGFR-dependent tumors. Sym004 is a recombinant antibody product consisting of a 1:1 mixture of two chimeric IgG1 mAb's targeting non-overlapping epitopes of EGFR. Preclinical proof-of-concept studies have shown that the Sym004 mixture is superior to existing anti-EGFR mAb's currently in clinical use (1). The nonclinical safety data of Sym004 are presented. The studies were designed to evaluate toxicity and pharmacokinetics of Sym004 as well as the individual antibodies comprising Sym004. No apparent accumulation was observed upon weekly dosing of Sym004 to non-human primates for eight consecutive weeks, whereas, a prolonged half life and accumulation were noted for both of the individual antibodies if these were administered alone. Weekly administration of Sym004 to non-human primates was well tolerated although dose limiting toxicity was observed in a dose-range-finding toxicology study. In a head-to-head comparison with the licensed anti-EGFR mAb, cetuximab (Erbitux®), weekly administration of Sym004 did not induce any distinct or novel adverse findings in the animals. However, and in accordance with the enhanced pharmacological profile of Sym004, an accelerated onset of anticipated anti-EGFR-mediated effects (skin rash and liquid faeces) was observed compared to cetuximab. None or only minor effects were observed in the animals treated with the individual antibodies comprising Sym004. All experimental observations, except a single case of rash, showed reversibility during the 4-week treatment free recovery period. In tissue cross reactivity studies, Sym004 had a staining pattern similar to cetuximab in a full tissue panel from both human and non human primates. In conclusion, Sym004 is considered to be safe, and the nonclinical safety data supported the initiation of the currently ongoing Phase I/II trial. 1. Pedersen MW et al. Cancer Res; 70(2) January 15, 2010 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1752. doi:10.1158/1538-7445.AM2011-1752

Abstract 3619: Tolerability in mice, monkeys, and rabbits of new antibody (MAb)-drug (SN-38) immunoconjugates

Abstract Antibody drug conjugates (ADCs) of the topoisomerase I inhibitor, SN-38, that allowed for a slow release of SN-38 from the ADC over time, were prepared using anti-CEACAM5 MAb, labetuzumab (hMN-14), anti-TROP-2 MAb, hRS7, and other MAbs by thiol-maleimide conjugation chemistry. These were shown previously to have significant anti-tumor activity in human cancer-xenograft models at cumulative doses frequently ≤ 100 mg/kg (≤ 8 mg/kg human equivalent dose; HED) without toxicity. To determine tolerability and dose-limiting toxicity, hRS7-SN-38 was examined in Swiss-Webster (S-W) mice and Cynomolgus monkeys (CMs), and labetuzumab-SN-38 was evaluated in New Zealand White rabbits. CMs do not express CEACAM5, but similarity in tissue cross-reactivity of hRS7 renders toxicity evaluation of hRS7-SN-38 conjugates in CM feasible. Male S-W mice (n = 14; ∼ 40 g) were administered i.p with 0 (control), 250, 500, or 750 mg/kg (0, 4, 8, and 12 mg/kg SN-38 equivalents) of hRS7-SN-38 on days 1 and 4. Half of the animals were necropsied 7 d, and the remainder 15 d, after the last dose. Blood counts, serum chemistry, and histopathology were performed. This ADC was also administered to CMs (3 M + 3 F/group), via i.v. infusion, at 0, 60, or 120 mg/kg (0, 0.96 and 1.92 mg/kg SN-38 equivalents, respectively) on days 1 and 4. Two males and 2 females from each group were euthanized and necropsied day 11, while the remaining animals were terminated on day 32. Blood counts and chemistries were collected pre-study and periodically through termination. The labetuzumab-SN-38 conjugate was examined in rabbits (n = 6-10) at 0, 30, 60, and 90 mg/kg, administered on days 1 and 4. Mice experienced no hematopoietic toxicity, and only transient weight loss of &amp;lt;10%, but there was a transient elevation in ALT and AST 7 days after treatment, which resolved in most animals one week later. There was no evidence of organ damage. Thus, mice tolerated a cumulative dose of 1500 mg/kg (HED ∼120 mg/kg) of the ADC. CMs receiving 2×60 mg/kg (HED = 2×20 mg/kg) had &amp;lt;7.3% weight loss, showed no abnormal serum chemistries, and while blood counts decreased, they remained within normal limits. Histopathological changes in the terminal group ranged from mild to moderate, but trended to normalcy in the recovery group. The 2×120 mg/kg (HED = 2×40 mg/kg) dose resulted in severe GI and hematologic toxicity. Rabbits tolerated labetuzumab-SN-38 conjugate at all the doses (HED up to 2×30 mg/kg), with uneventful histopathology findings. Pharmacokinetic analysis of rabbit serum showed the IgG cleared with a half-life of ∼ 2 d, and SN-38 clearance was consistent with in vitro stability of the ADC in rabbit serum. ADC and free SN-38 were found to have similar mechanisms of action in vitro. These findings, coupled with previous efficacy studies in xenograft models, indicate a favorable therapeutic window for these ADCs and support the clinical evaluation of these agents in their appropriate indications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3619. doi:10.1158/1538-7445.AM2011-3619

Survey results on the use of the tissue cross-reactivity immunohistochemistry assay

A multinational pharmaceutical and biotechnology company survey was conducted to gain a better understanding of the use and value of the tissue cross-reactivity (TCR) assay in the development of biotherapeutic molecules. The majority of the molecules did not use TCR data as the only basis for determining species selection for toxicity studies (73%). For 95% of the molecules, the TCR data had no impact on the development strategy. For 2% of the molecules (1/56), TCR data was the sole source of information indicating a potential risk to patients. Unexpected or off-target binding was seen with 35% of the molecules, with the majority of this binding occurring in the CNS and reproductive organs. Tissues that were known or presumed to contain the target stained positively in 22% and 10% of molecules tested in non-human primate and human tissues, respectively. Tissues that were known or presumed to lack the target were negative for staining in 39% and 50% of molecules for non-human primate and human tissue, respectively. For 5% (6/110) of all the molecules, companies stated that toxicities would have been missed in animal studies or the clinic (i.e., not identified by clinical signs, histopathology, etc.) if the TCR studies had not been performed.

Use of Tissue Cross-reactivity Studies in the Development of Antibody-based Biopharmaceuticals

Tissue cross-reactivity (TCR) studies are screening assays recommended for antibody and antibody-like molecules that contain a complementarity-determining region (CDR), primarily to identify off-target binding and, secondarily, to identify sites of on-target binding that were not previously identified. At the present time, TCR studies involve the ex vivo immunohistochemical (IHC) staining of a panel of frozen tissues from humans and animals, are conducted prior to dosing humans, and results are filed with the initial IND/CTA to support first-in-human clinical trials. In some cases, a robust TCR assay cannot be developed, and in these cases the lack of a TCR assay should not prevent a program from moving forward. The TCR assay by itself has variable correlation with toxicity or efficacy. Therefore, any findings of interest should be further evaluated and interpreted in the context of the overall pharmacology and safety assessment data package. TCR studies are generally not recommended for surrogate molecules or for comparability assessments in the context of manufacturing/cell line changes. Overall, the design, implementation, and interpretation of TCR studies should follow a case-by-case approach.

The importance of characterization of FITC-labeled antibodies used in tissue cross-reactivity studies

Fluorescein isothiocyanate (FITC)-labeled antibodies are widely used as primary antibodies in the tissue cross-reactivity (TCR) studies for the development of therapeutic antibodies. However, the effects of FITC-labeling on the characteristics of an antibody are poorly understood. The present study was performed to examine the effect of FITC-labeling on the binding affinity and immunohistochemical staining profile of an antibody, using several antibodies with different FITC-labeling indices. The results showed that the FITC-labeling index in antibody was negatively correlated with the binding affinity for its target antigen. Immunohistochemically, an antibody with a higher labeling index had a tendency to be more sensitive, but was also more likely to yield non-specific staining. Based on these findings, we recommend that a FITC-labeled antibody used as a primary antibody in a TCR study should be carefully selected from several differently labeled antibodies to minimize the decrease in the binding affinity and achieve the appropriate sensitivity and interpretation of the immunohistochemistry.

Phase I dose-escalation study of AGS-1C4D4, an anti-PSCA human antibody in castration-resistant prostate cancer (CRPC).

4669 Background: AGS-1C4D4 is a fully human MAb directed to PSCA, a cell surface antigen expressed on tumor cells in the majority of patients with prostate, pancreatic and bladder cancers. AGS-1C4D4 is produced in CHO cells and has an identical sequence to that of the hybridoma-derived AGS-PSCA. Preclinical studies confirmed comparability to AGS-PSCA in tumor inhibition tissue cross reactivity and PK. Due to differences in glycosylation and ADCC in vitro, a limited AGS-1C4D4 phase I trial was undertaken to explore potential differences in safety and PK. Methods: Thirteen patients with metastatic CRPC were treated. AGS-1C4D4 was administered by IV infusion Q3W for 4 doses at 6, 12, 24, and 48 mg/kg. Primary endpoints were safety and PK. Secondary endpoints included immunogenicity and anti-tumor activity. Disease assessments were performed Q12W. Patients with stable disease (SD) were eligible to receive extended treatment at the same dose and schedule. Results: Nine of 13 patients completed 4 doses. Toxicities were primarily grade 1-2. No deaths or grade 3-4 treatment-related events, acute infusion-related toxicities, or anti AGS-1C4D4 antibodies have been reported. PK was linear. Similarly to AGS-PSCA, serum concentrations of AGS-1C4D4 declined biphasically and the final elimination Ph was characterized by a slow clearance rate (CL) and a long terminal T1/2. Median CL values for the 4 dose levels ranged from 0.10 to 0.14 mL/h-kg; and T1/2 was from 2.2 to 2.9 weeks. The target trough serum antibody concentration of 200 mg/ml, based on xenograft models, was reached in most of the higher dose patients with available PK data and was maintained with subsequent dosing. SD was reported in 5 (38%) patients and all these received extended dosing with total treatment duration of up to 36 weeks (median 24, range 18-36). No PSA responses were observed. Conclusions: AGS-1C4D4 was well tolerated. PK indicated slow clearance and long T1/2 similar to AGS-PSCA. Radiographic SD was noted in 38% of the patients. Despite preclinical differences in ADCC in vitro, this phase I study confirmed similar safety and PK to the hybridroma-derived AGS-PSCA. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Agensys Astellas Pharma Agensys

MDX-1097 Binds Specifically to Kappa Myeloma Cells and Anti-Tumour Activity Is Mediated by Multiple Effector Cells.

Abstract 1846Poster Board I-872Previous studies have described a murine monoclonal antibody, mKap, that specifically recognizes a cell surface antigen expressed on kappa myeloma cells and not on normal lymphoid cells. This antigen has been identified and designated kappa myeloma antigen (KMA). KMA consists of free kappa light chains (kFLC) not associated with heavy chain and is present on plasma cells isolated from kappa myeloma (MMk) patient bone marrow aspirates, kappa myeloma cell lines and kappa macroglobulinemia. In vitro data demonstrated that mKap was able to inhibit cell growth and induce apoptosis in myeloma cell lines. In addition, pre-clinical studies demonstrated that mKap was well tolerated and showed significant efficacy in a SCID xenograft model of MM.MDX-1097 is a chimeric version of mKap that is currently in development for the treatment of kappa restricted multiple myeloma. The antibody retains the binding affinity and specificity of mKap. Specific binding of MDX-1097 to malignant plasma cells isolated from MMk patient bone marrow aspirates has recently been demonstrated by flow cytometry. In addition a human tissue cross-reactivity study was performed using immunohistochemistry to assess the potential binding of MDX-1097-FITC to cryosections taken from a human tissue panel of three normal donors. The results demonstrated that MDX-1097 bound to bone marrow plasma cells from two patients with kappa cell dyscrasia but did not bind to normal human tissue samples or to plasma cells from a patient with lambda plasmacytoma. The ability of serum kFLC to inhibit MDX-1097 binding to the myeloma cell line, JJN3, was assessed by flow cytometry using serum derived from 32 MMk patients. The results indicated that MDX-1097 at a concentration of 100μg/mL (equivalent to an estimated serum concentration of 5mg/kg dose) is capable of binding to myeloma cells in the presence of 0–250μg/mL of serum kFLC. In vitro functional studies have demonstrated that MDX-1097 engages Fc receptor bearing effector cells and induces antibody dependent cellular cytotoxicity (ADCC) in kappa myeloma cell lines in the presence of healthy donor peripheral blood mononuclear cells. Further investigations have verified that purified natural killer cells (NK) play a major role in MDX-1097 anti-tumour activity. Importantly, recent studies have demonstrated that antibody dependent cellular phagocytosis by macrophages contributes to the anti-tumour activity of several therapeutic monoclonal antibodies. Preliminary data indicates that MDX-1097 may be capable of inducing enhanced uptake by macrophages.In conclusion MDX-1097 showed specific binding to KMA on myeloma cells isolated from patient's bone marrow samples and antibody binding is observed in the presence of kFLC in patient serum. In addition MDX-1097 anti-tumour activity is probably mediated by multiple Fc receptor bearing effector cells. Disclosures:Wong:Immune System Therapeutics: Employment. Asvadi:Immune System Therapeutics: Employment. Dunn:Immune System Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Jones:Immune System Therapeutics: Employment. Campbell:Immune System Therapeutics: Employment.

Validation of tissue cross reactivity (TCR) studies: Tissue cross reactivity of anti-human CD209 in cynomolgus tissues

Validation of tissue cross reactivity (TCR) studies: Tissue cross reactivity of anti-human CD209 in cynomolgus tissues

Evaluation of a surrogate antibody for preclinical safety testing of an anti-CD11a monoclonal antibody

Surrogate antibodies are a potential solution to the limited safety testing possible with humanized monoclonal antibodies with restricted species cross-reactivity. However, there are currently no defined criteria by which a potential surrogate antibody should be judged prior to its use in determining safety issues for the clinical agent. We propose that, potential surrogates should undergo rigorous evaluation to assess pharmacological and toxicological activities in comparison to the clinical agent. The current studies evaluated a chimeric mouse/rat anti-mouse CD11a monoclonal antibody (muM17) as a potential surrogate for efalizumab, a humanized anti-CD11a antibody in development for psoriasis. CD11a is a subunit of lymphocyte function associated antigen-1, an integrin involved in cell–cell interactions important to immune responses and inflammation. In vitro pharmacology studies included binding affinity to whole mouse blood and inhibitory activity of muM17 in a mixed lymphocyte response assay. In vivo pharmacology was examined using a delayed type hypersensitivity assay in female CD-1 mice. The toxicology evaluation included a murine tissue cross-reactivity study and in vivo multiple dose studies in female CD-1 mice which were administered muM17 (0.1–30 mg/kg) via subcutaneous injections once a week for 4 weeks. Clinical observations, body weight, clinical pathology, T cell CD11a expression, immunogenicity, toxicokinetics, and lymphoid organ histopathology were evaluated. Finally, since reproductive safety testing would be an important application of the proposed surrogate antibody, a pilot study in pregnant mice was conducted that demonstrated proportional transfer of muM17 into the fetus. These studies demonstrated that muM17 has pharmacological and toxicological activities similar to efalizumab. The selection of dose and regimen for GLP (Good Laboratory Practice) toxicology studies and extrapolation to clinical dose levels was based on pharmacodynamic activity (CD11a downmodulation on T cells).