Anaphylaxis caused by repetitive doses of a GITR agonist monoclonal antibody in mice.

Immunotherapy for cancer using antibodies to enhance T-cell function has been successful in recent clinical trials. Many molecules that improve activation and effector function of T cells have been investigated as potential new targets for immunomodulatory antibodies, including the tumor necrosis factor receptor superfamily members GITR and OX40. Antibodies engaging GITR or OX40 result in significant tumor protection in preclinical models. In this study, we observed that the GITR agonist antibody DTA-1 causes anaphylaxis in mice upon repeated intraperitoneal dosing. DTA-1-induced anaphylaxis requires GITR, CD4(+) T cells, B cells, and interleukin-4. Transfer of serum antibodies from DTA-1-treated mice, which contain high levels of DTA-1-specific immunoglobulin G1 (IgG1), can induce anaphylaxis in naive mice upon administration of an additional dose of DTA-1, suggesting that anaphylaxis results from anti-DTA-1 antibodies. Depletion of basophils and blockade of platelet-activating factor, the key components of the IgG1 pathway of anaphylaxis, rescues the mice from DTA-1-induced anaphylaxis. These results demonstrate a previously undescribed lethal side effect of repetitive doses of an agonist immunomodulatory antibody as well as insight into the mechanism of toxicity, which may offer a means of preventing adverse effects in future clinical trials using anti-GITR or other agonist antibodies as immunotherapies.

Preclinical Efficacy of the Antibody-Drug Conjugate CLDN6-23-ADC for the Treatment of CLDN6-Positive Solid Tumors.

PURPOSE: Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody-drug conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6-23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker. EXPERIMENTAL DESIGN: A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6-23-ADC. The antitumor efficacy of CLDN6-23-ADC was assessed for antitumor efficacy in CLDN6-positive (CLDN6+) and -negative (CLDN6-) xenografts and patient-derived xenograft (PDX) models of human cancers. RESULTS: CLDN6-23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells in vitro, and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition led to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6-23-ADC. IHC assessment of cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target. CONCLUSIONS: We report the development of a novel ADC, CLDN6-23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6-23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing phase I study.

Development of a Novel CLDN18.2-directed Monoclonal Antibody and Antibody-Drug Conjugate for Treatment of CLDN18.2-Positive Cancers.

Gastric and pancreatic cancers are malignancies of high unmet clinical need. Expression of CLDN18.2 in these cancers, coupled with it's absence from most normal tissues, provides a potential therapeutic window against this target. We present preclinical development and characterization of a novel therapeutic mAb and antibody-drug conjugate (ADC) targeting CLDN18.2. A humanized CLDN18.2 specific mAb, CLDN18.2-307-mAb, was generated through immunization in mice followed by full humanization of the mouse mAb sequences. Antibody clones were screened by flow cytometry for selective binding to membrane bound CLDN18.2. A CLDN18.2-directed ADC (CLDN18.2-307-ADC) was also generated by conjugating MMAE to CLDN18.2 mAb using a cleavable linker. Tissue expression of CLDN18.2 was determined by IHC assay using a CLDN18.2-specific mAb. CLDN18.2-307-mAb binds with high affinity to CLDN18.2-positive (CLDN18.2+) cells and induces antibody-dependent cell-mediated cytotoxicity (ADCC). Treatment with this CLDN18.2-mAb blocked the growth of CLDN18.2+ gastric and pancreas cancer cell line xenograft (CDX) models. Upon binding to the extracellular domain of this target, the CLDN18.2-ADC/CLDN18.2 protein was internalized and subsequently localized to the lysosomal compartment inducing complete and sustained tumor regressions in CLDN18.2+ CDXs and patient-derived pancreatic cancer xenografts (PDX). A screen of human cancer tissues, by IHC, found 58% of gastric, 60% of gastroesophageal junction, and 20% of pancreatic adenocarcinomas to be positive for membrane expression of CLDN18.2. These data support clinical development of the CLDN18.2-307-mAb and CLDN18.2-307-ADC for treatment of CLDN18.2+ cancers. Both are now being investigated in phase I clinical studies.

An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

TCR mimic compounds for pHLA targeting with high potency modalities in oncology.

pHLA complexes represent the largest class of cell surface markers on cancer cells, making them attractive for targeted cancer therapies. Adoptive cell therapies expressing TCRs that recognize tumor specific pHLAs take advantage of the unique selectivity and avidity of TCR: pHLA interactions. More recently, additional protein binding domains binding to pHLAs, known as TCR mimics (TCRm), were developed for tumor targeting of high potency therapeutic modalities, including bispecifics, ADCs, CAR T and -NK cells. TCRm compounds take advantage of the exquisite tumor specificity of certain pHLA targets, including cell lineage commitment markers and cancer testis antigens (CTAs). To achieve meaningful anti-tumor responses, it is critical that TCRm compounds integrate both, high target binding affinities and a high degree of target specificity. In this review, we describe the most advanced approaches to achieve both criteria, including affinity- and specificity engineering of TCRs, antibodies and alternative protein scaffolds. We also discuss the status of current TCRm based therapeutics developed in the clinic, key challenges, and emerging trends to improve treatment options for cancer patients treated with TCRm based therapeutics in Oncology.

Molecular engineering and design of therapeutic antibodies.

Since the first murine monoclonal antibody was approved for human therapeutic use over a decade ago, the realization that monoclonal antibody therapeutics could be engineered to improve their efficacy has inspired an astonishing array of novel antibody constructs. Early focus was on reducing the immunogenicity of rodent antibodies via humanization and generation of antibodies in transgenic mice; as those techniques were being established and then provided marketed therapeutic antibodies, the focus expanded to include engineering for enhanced effector functions, control of half-life, tumor and tissue accessibility, augmented biophysical characteristics such as stability, and more efficient (and less costly) production. Over the past two years significant progress in designing antibodies with improved pharmacokinetic properties, via modified interaction with the neonatal Fc receptor (FcRn), has been achieved. Likewise, the ability to alter the communication of a therapeutic antibody with the immune system has been advanced, using both manipulation of the immunoglobulin protein sequence and its glycosylation. Although clinical evaluation of these engineered modifications has yet to be reported, results in primates are encouraging.

Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis.

IL-23 is a member of the IL-12 cytokine family that drives a highly pathogenic T cell population involved in the initiation of autoimmune diseases. We have shown that IL-23-dependent, pathogenic T cells produced IL-17 A, IL-17 F, IL-6, and TNF but not IFN-gamma or IL-4. We now show that T-bet and STAT1 transcription factors are not required for the initial production of IL-17. However, optimal IL-17 production in response to IL-23 stimulation appears to require the presence of T-bet. To explore the clinical efficacy of targeting the IL-23 immune pathway, we generated anti-IL-23p19-specific antibodies and tested to determine whether blocking IL-23 function can inhibit EAE, a preclinical animal model of human multiple sclerosis. Anti-IL-23p19 treatment reduced the serum level of IL-17 as well as CNS expression of IFN-gamma, IP-10, IL-17, IL-6, and TNF mRNA. In addition, therapeutic treatment with anti-IL-23p19 during active disease inhibited proteolipid protein (PLP) epitope spreading and prevented subsequent disease relapse. Thus, therapeutic targeting of IL-23 effectively inhibited multiple inflammatory pathways that are critical for driving CNS autoimmune inflammation.

Antibody engineering for therapeutics.

With the acceptance of antibodies as therapeutics, a diversity of engineered antibody forms have been created to improve their efficacy, including enhancing the effector functions of full-length antibodies, delivering toxins to kill cells or cytokines in order to stimulate the immune system, and bispecific antibodies to target multiple receptors. After years of in vitro investigation, many of these are now moving into clinical trials and are showing promise. A potential new type of effector function for antibodies, that is, the generation of reactive oxygen species that may effect inflammation or bacterial killing, has been elucidated. In addition, the field has expanded beyond a concentration on immunoglobulin G to include immunoglobulin A antibodies as potential therapeutics.

Engineering of therapeutic antibodies to minimize immunogenicity and optimize function.

One of the first difficulties in developing monoclonal antibody therapeutics was the recognition that human anti-mouse antibody (HAMA) response limited the administration of murine antibodies. Creative science has lead to a number of ways to counter the immunogenicity of non-human antibodies, primarily through chimeric, humanized, de-immunized, and most recently, human-sequence therapeutic antibodies. Once therapeutic antibodies of low or no immunogenicity were available, the creativity then turned to engineering both the antigen-binding domains (e.g., affinity maturation, stability) and altering the effector functions (e.g. antibody-dependent cellular cytotoxicity, complement-dependent cellular cytotoxicity, and clearance rate).

Inhibition of insulin-like growth factor-I receptor (IGF-IR) signaling and tumor cell growth by a fully human neutralizing anti-IGF-IR antibody.

Insulin-like growth factor-I receptor (IGF-IR) plays an important role in tumor cell growth and survival. On ligand stimulation, IGF-IR, a receptor tyrosine kinase, phosphorylates tyrosine residues on two major substrates, IRS-1 and Shc, which subsequently signal through the Ras/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT pathways. Here, we describe the characterization of a fully human anti-IGF-IR monoclonal antibody 19D12 that inhibits IGF binding and autophosphorylation of both IGF-IR/IGF-IR homodimers and IGF-IR/insulin receptor heterodimers. 19D12 does not recognize insulin receptor homodimers. In addition to inhibiting IGF-IR autophosphorylation, 19D12 also inhibits IRS-1 phosphorylation and activation of the major downstream signaling molecules AKT and extracellular signal-regulated kinase 1/2. Furthermore, the antibody down-regulates the total IGF-IR protein level and can exhibit antibody-dependent cellular cytotoxicity activity against a non-small cell adenocarcinoma cell line in vitro in the presence of isolated human natural killer cells. 19D12 binds tightly to the receptor, with an affinity of 3.8 pmol/L as measured by KinExA. In cell culture, 19D12 inhibits proliferation and soft agar growth of various tumor cell lines. In vivo, 19D12 inhibits the tumor growth of a very aggressive human ovarian tumor xenograft model A2780. These data support the development of this anti-IGF-IR monoclonal antibody as a promising anticancer agent.

Structural insight into how an anti-idiotypic antibody against D3H44 (anti-tissue factor antibody) restores normal coagulation.

6A6 is a murine monoclonal antibody raised against the humanized anti-tissue factor antibody D3H44. 6A6 is able to completely neutralize the anticoagulant activity of D3H44 in tissue factor-dependent functional assays, such as endotoxin-induced whole blood clotting, prothrombin time, as well as factor X and factor IX activation. ELISA-type assays further showed that 6A6 binds to an epitope with critical determinants on the V(L) domain of D3H44. The possibility that the anti-idiotypic 6A6 might carry an "internal image" of the original antigen (tissue factor) was examined using the X-ray structure of the 6A6-Fab/D3H44-Fab complex determined at 2.5A resolution. We find that 6A6 structurally mimics tissue factor only so far as it combines with the antigen recognition surface of D3H44. While 6A6 contacts both V(L) and V(H) domains of D3H44, as does tissue factor, there is more contact with the D3H44 V(L) domain and less with the D3H44 V(H) domain relative to the tissue factor contacts on D3H44. Additionally, there is an almost total lack of correspondence between 6A6 and tissue factor at the level of amino acid side-chain functional groups. Despite the fact that both tissue factor and 6A6 are composed largely of beta-sheets, they present fundamentally different elements of secondary structure to D3H44; tissue factor presents beta-sheets edge-on, while 6A6 uses mostly loops. Finally, the finding that 6A6 competes with tissue factor for D3H44 binding raises the possibility of using 6A6 as an antidote for D3H44 anticoagulant therapy. To this end, we constructed a chimeric murine/human 6A6-Fab, which effectively neutralized D3H44 and fully restored tissue factor function in enzymatic assays.

Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis.

Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.

Simple quantitative live cell and anti-idiotypic antibody based ELISA for humanized antibody directed to cell surface protein CD20.

Rituxan, a chimeric anti-CD20 antibody, has been used for treating non-Hodgkin's lymphoma and some autoimmune diseases. However, a humanized anti-CD20 antibody is desirable for long-term treatment of autoimmune diseases. CD20 is an integral membrane protein with a small intervening extracellular loop. Lacking a native soluble CD20 protein, we developed a simple cell-based enzyme-linked immunosorbent assay (ELISA) using live WIL2 cells in a 96-well format to measure relative binding affinity to support the humanization process. Although WIL2 cells grow in suspension and require centrifugation during the wash steps, the assay was quantitative and reproducible. We also demonstrated that cloned adherent transfected Chinese hamster ovary (CHO) cells could be used to improve assay throughput. For clinical studies requiring quantification of the humanized antibody in serum, we used an alternate approach and developed a high throughput ELISA using an anti-idiotypic antibody as a surrogate antigen for capture and an anti-idiotypic antibody for detection to overcome serum effects. These assay strategies may be applied for characterization of other antibodies directed to multitransmembrane proteins.

Engineering antibodies for therapy.

With eleven therapeutic antibodies approved worldwide and many more in clinical trials, research on antibody engineering has continued to escalate and expand. This review covers recent progress in generation of antibodies by ex vivo methods, systems for screening these, and the quest for higher affinity, more stable, optimally biodistributed antibody fragments, especially for solid tumors. The latest developments in engineering antibodies for removal or enhancement of effector functions (antibody-dependent cellular cytotoxicity (ADCC), phagosytosis, complement fixation (CDC) and half-life) through protein alteration or carbohydrate optimization may now enable generation of superior antibody therapeutics. Antibody conjugates, including immunotoxins and immunocytokines, as well as multivalent and multispecific antibodies confer expanded utility of therapeutic antibodies. Finally, research into the IgA/FcalphaRI system has now provided an additional route to therapeutic antibodies.

Identification of IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn: comparison of soluble receptor-based and cell-based binding assays.

Clinical response to the anti-CD20 antibody rituximab has been demonstrated to correlate with the polymorphism in the FcγRIIIa receptor where patients homozygous for the higher affinity V158 allotype showed a better response rate. This finding suggests that engineering of anti-CD20 for increased FcγRIIIa affinity could result in improved clinical outcome. To identify variants with increased affinity to FcγRIIIa, we developed quantitative assays using soluble receptors as well as engineered cell lines expressing FcγRI or FcγRIIIa on the cell surface. We assayed a set of anti-CD20 IgG(1) variants that had identical Fab regions, but alterations in the Fc regions, in both the soluble receptor-based and cell-based FcγRIIIa binding assays. We obtained similar relative binding affinity increases and assay precisions. The increase in affinity for FcγRIIIa correlated with the increase in activity in the antibody-dependent cellular cytotoxicity assay. These variants had unaltered FcγRI binding. In addition to Fcγ receptors, IgG also binds to FcRn, the receptor responsible for the long circulating half-life of IgG. The mutations in the anti-CD20 variants were previously found not to affect FcRn binding in the soluble receptor-based assays; consequently, we used anti-Her2 variants with different binding affinities to FcRn to study FcRn binding assays. We generated a cell line expressing FcRn on the cell surface to measure IgG binding and obtained similar ranking of these anti-Her2 variants in the cell-based and the soluble receptor-based FcRn binding assays. In conclusion, both the soluble receptor-based and cell-based binding assays can be used to identify IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn.

Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab.

Dimerization is essential for activity of human epidermal growth factor receptors (HER1/EGFR, HER2/ErbB2, HER3/ErbB3, and ErbB4) and mediates intracellular signaling events leading to cancer cell proliferation, survival, and resistance to therapy. HER2 is the preferred dimerization partner. Activation of HER signaling pathways may be blocked by inhibition of dimer formation using a monoclonal antibody (MAb) directed against the dimerization domain of HER2. The murine MAb 2C4 that specifically binds the HER2 dimerization domain was cloned as a chimeric antibody, humanized using a computer-generated model to guide framework substitutions, and variants were tested as Fabs. Pharmacokinetics and toxicology were evaluated in rodents and cynomolgus monkeys. Cloning the variable domains of MAb 2C4 into a vector containing human kappa and CH1 domains allowed construction of a mouse-human chimeric Fab. DNA sequencing of the chimeric clone permitted identification of CDR residues. The full-length IgG1 of variant F-10 was equivalent in binding to chimeric IgG1 and was designated pertuzumab (rhuMAb 2C4; Omnitarg). Pertuzumab pharmacokinetics was best described by a two-compartment model with a distribution phase of <1 day, terminal half-life of approximately 10 days, and volume of distribution of approximately 40 mL/kg that approximates serum volume. With the exception of diarrhea, pertuzumab was generally well tolerated in cynomolgus monkeys. Pertuzumab, a recombinant humanized IgG1 MAb, is the first of a new class of agents known as HER dimerization inhibitors. Inhibition of HER dimerization may be an effective anticancer strategy in tumors with either normal or elevated expression of HER2.

Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease.

The MHC class I-like Fc receptor FcRn plays an essential role in extending the half-life (t(1/2)) of IgG antibodies and IgG-Fc-based therapeutics in the circulation. The goal of this study was to analyze the effect of human IgG1 (hIgG1) antibodies with enhanced in vitro binding to FcRn on their in vivo t(1/2) in mice expressing human FcRn (hFcRn). Mutants of the humanized monoclonal Herceptin antibody (Hu4D5-IgG1), directed against human epidermal growth factor receptor 2 (p185 (HER2)), show altered pH-dependent binding to hFcRn in vitro. Two engineered IgG1 mutants (N434A and T307A/E380A/N434A) showed a considerably extended t(1/2) in vivo compared with wild-type antibody in mice expressing an hFcRn transgene (Tg) but not in mice expressing the endogenous mouse FcRn. The efficiency of hFcRn-mediated protection was dependent on hFcRn Tg copy number. Moreover, when injected into FcRn-humanized mice at a concentration sufficient to partially saturate hFcRn, the engineered IgG1 mutants with an extended serum t(1/2) were most effective in reducing the t(1/2) of a tracer hIgG1 antibody. Finally, administration of mutant with high binding to hFcRn ameliorated arthritis induced by passive transfer with human pathogenic plasma. These results indicate that Fc regions modified for high binding affinity to hFcRn increases serum persistence of therapeutic antibodies, that the same approach can be exploited as an anti-autoimmune therapy to promote the clearance of endogenous pathogenic IgG and that FcRn-humanized mice are a promising surrogate for hIgG therapeutic development.

Selection, design, and engineering of therapeutic antibodies.

mAbs account for an increasing portion of marketed human biological therapeutics. As a consequence, the importance of optimal selection, design, and engineering of these not only has expanded in the past 2 decades but also is now coming into play as a competitive factor. This review delineates the 4 basic areas for optimal therapeutic antibody selection and provides examples of the increasing number of considerations necessary for, and options available for, antibody design. Though some of the advances in antibody technology (eg, antibodies derived from phage-display libraries) have already made it to market, other more recent advances, such as engineering antibodies for enhanced effector functions, may not be far behind, especially given the increasing competition for therapeutic antibodies to the same target (eg, anti-CD20 and anti-TNF-alpha).