Targeting and pharmacology of an anti-IL13Rα2 antibody and antibody-drug conjugate in a melanoma xenograft model.

IL13Rα2 is a cell surface tumor antigen that is overexpressed in multiple tumor types. Here, we studied biodistribution and targeting potential of an anti-IL13Rα2 antibody (Ab) and anti-tumor activity of anti-IL13Rα2-antibody-drug conjugate (ADC). The anti-IL13Rα2 Ab was labeled with fluorophore AF680 or radioisotope 89Zr for in vivo tracking using fluorescence molecular tomography (FMT) or positron emission tomography (PET) imaging, respectively. Both imaging modalities showed that the tumor was the major uptake site for anti-IL13Rα2-Ab, with peak uptake of 5-8% ID and 10% ID/g as quantified from FMT and PET, respectively. Pharmacological in vivo competition with excess of unlabeled anti-IL13Rα2-Ab significantly reduced the tumor uptake, indicative of antigen-specific tumor accumulation. Further, FMT imaging demonstrated similar biodistribution and pharmacokinetic profiles of an auristatin-conjugated anti-IL13Rα2-ADC as compared to the parental Ab. Finally, the anti-IL13Rα2-ADC exhibited a dose-dependent anti-tumor effect on A375 xenografts, with 90% complete responders at a dose of 3 mg/kg. Taken together, both FMT and PET showed a favorable biodistribution profile for anti-IL13Rα2-Ab/ADC, along with antigen-specific tumor targeting and excellent therapeutic efficacy in the A375 xenograft model. This work shows the great potential of this anti-IL13Rα2-ADC as a targeted anti-cancer agent.

Discovery of A Novel EGFR-Targeting Antibody-Drug Conjugate, SHR-A1307, for the Treatment of Solid Tumors Resistant or Refractory to Anti-EGFR Therapies.

Although inhibiting EGFR-mediated signaling proved to be effective in treating certain types of cancers, a quickly evolved mechanism that either restores the EGFR signaling or activates an alternative pathway for driving the proliferation and survival of malignant cells limits the efficacy and utility of the approach via suppressing the EGFR functionality. Given the fact that overexpression of EGFR is commonly seen in many cancers, an EGFR-targeting antibody-drug conjugate (ADC) can selectively kill cancer cells independently of blocking EGFR-mediated signaling. Herein, we describe SHR-A1307, a novel anti-EGFR ADC, generated from an anti-EGFR antibody with prolonged half-life, and conjugated with a proprietary toxin payload that has increased index of EGFR targeting-dependent versus EGFR targeting-independent cytotoxicity. SHR-A1307 demonstrated strong and sustained antitumor activities in EGFR-positive tumors harboring different oncogenic mutations on EGFR, KRAS, or PIK3CA. Antitumor efficacy of SHR-A1307 correlated with EGFR expression levels in vitro and in vivo, regardless of the mutation status of EGFR signaling mediators and a resultant resistance to EGFR signaling inhibitors. Cynomolgus monkey toxicology study showed that SHR-A1307 is well tolerated with a wide therapeutic index. SHR-A1307 is a promising therapeutic option for EGFR-expressing cancers, including those resistant or refractory to the EGFR pathway inhibitors.

Generation of therapeutic immunoconjugates via Residue-Specific Conjugation Technology (RESPECT) utilizing a native cysteine in the light chain framework of Oryctolagus cuniculus.

The conjugation of toxins, dyes, peptides, or proteins to monoclonal antibodies is often performed via free thiol groups generated by either partial reduction methods or engineering free cysteine residues into the antibody sequence. Antibodies from the rabbit Oryctolagus cuniculus have an additional intrachain disulfide bond, whereby the light chain variable kappa domain is bridged to the constant kappa region between cysteine residues at positions 80 and 171, respectively. Chimerization of rabbit antibodies with human constant domains allows for the generation of a free thiol group at the light chain position 80 (C80) that can be used for site-specific conjugation. An efficient process for the purification and simultaneous removal of cysteinylation at the C80 site was developed. The unpaired C80 was shown to be efficiently conjugated using several different maleimido-based ligands. REsidue SPEcific Conjugation Technology (RESPECT) antibody-drug conjugates prepared using rabbit-human chimeric anti-human mesothelin rabbit antibodies and maleimido-PEG2-auristatin conjugated to C80 were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model.

Preclinical Antitumor Efficacy of BAY 1129980-a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer.

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non-small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody-drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893-904. ©2017 AACR.

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.

[Immunodepressive properties of antineoplastic agents]. / Immunodepressivnye svoistva nekotorykh protivoopukholevykh preparatov.

Experiments on CBA mice immunized with sheep red blood cells have shown that paphencyl, promycil, prospidin and imidazole-4-carboxamide decrease the number of IgM-antibody-forming cells in mouse spleens during the primary immune response. The highest immunodepressant effect was exhibited by paphencyl, while the least by prospidin. The maximum inhibition of the immune response was observed on paphencyl and promycil administration 24 hours after the immunization, that on prospidin administration 24 hours prior to antigen exposure, and that on imidazole-4-carboxamide administration 24 hours prior and 48 hours after the antigenic stimulation. The degree of antibody genesis suppression depends on the dose of paphencyl, promycil and prospidin and does not depend on the dose of imidazole-4-carboxamide. Paphencyl significantly diminishes the number of hemopoietic stem cells in mouse spleens, while prospidin was less active in this respect.

Cross immunological reactions between three haptens of the "para" group and 4-aminoantipyrine.

Cross passive hemagglutination reactions between sulfanilic acid (SA), para-aminobenzoic acid (PABA), p-phenethidine (PT) and 4-aminoantipyrine (4-AA) haptens have been investigated using conjugates of diazo derivaties of these compounds with homologous serum proteins. Intense cross passive hemagglutination reactions were found between PABA, SA and 4-AA haptens. Although anti-PT sera cross reacted intensely with PABA, SA as well as with 4-AA haptens, antibodies cross reacting with PT were not detected in any of the anti-PABA, anti-SA and anti-4-AA antisera. The conjugates containing a heterologous hapten de not substantially alter the titer of the hemagglutination reactions performed with erythrocytes coated with the homologous hapten, but completely suppress the hemagglutination reactions performed with erythrocytes coated with this hapten as well as with other, but not all, heterologous, haptens. The results support the conclusion that the apparent polysensitization to drugs may actually rely upon cross reactions with haptens chemically related to the immunizing one.

Induction of T cell response to haptens coupled to mycobacteria.

A new method for the induction of cellular immune response to commonly used haptens in the absence of detectable antibody response is described. Different haptens were convalently coupled to Mycobacteria and they were injected into guinea pigs in incomplete Freund's adjuvant. Humoral and cellular immune response to haptens were examined at weekly intervals for 5 weeks. Our results show that a significant anti-hapten cellular response was induced and subsequently elicited by both in vivo (skin test) and in vitro (Lymphocyte transformation and macrophage migration inhibition) assays.

Production of guinea pig IgG1 homocytotropic antibodies to hapten-conjugated homologous serum albumin with different adjuvant combinations.

The effects of various antigen-adjuvant combinations on the production of IgG1 antibody was investigated with p-aminobenzoate conjugated to guinea pig serum albumin. The results demonstrate the importance that initial exposure of antigen-adjuvant combination has on IgG1 production. Further, there is a synergistic effect when antigen adsorbed to alum and antigen with complete Freund's adjuvant were used in conjunction with one another. The result of this effect is discussed within the context of T and B cell activation by antigen-adjuvant combinations.

N-bromosuccinimide oxidation of anti-DNP and anti-DNP-p-aminobenzoylglutamate antibodies in the presence and absence of protecting hapten.

The reagent N-bromosuccinimide (NBS) has been employed to investigate the role of tryptophan in hapten binding in anti-DNP (H-1) and anti-DNP-p-aminobenzoylglutamate (DNP-ABG) (I-13) antibodies. In 0-1 M acetate (pH 4-0) buffer fifteen and sixteen moles of tryptophan in the anti-DNP and anti-DNP-ABG antibodies respectively were reactive toward NBS. The hapten DNP-lysine protected 1 tryptophan in antibody H-1 and three tryptophans in antibody I-13 from NBS modification. DNP-ABG protected three tryptophans in antibody H-1 and five tryptophans in antibody I-13 from NBS oxidation. NBS treatment of the unprotected antibodies resulted in a significant, but not total inhibition of hapten binding, while in the hapten protected antibody preparation no significant loss of binding occurred due to NBS treatment. The binding activity of the anti-DNP antibody H-1 was more sensitive to NBS oxidation than was the anti-DNAP-ABG antibody I-13. This was apparently due to the larger number of oxidizable tryptophans in I-13 making it less sensitive to overall tryptophan modification. The results of these investigations are discussed in terms of the antibody combining site model proposed by Haselkorn et al. (Haselkorn, Friedman, Givol and Pecht, 1974) derived from kinetic mapping of the antibody-combining site by chemical relaxation spectroscopy.

Comparison of specificities of humoral and cellular immune response to haptens.

In this study we induced humoral and cellular immunity to three isomers of aminosulfanilic and aminobenzoic acids and compared their respective specificities. These studies were facilated by using a new method for preferentially inducing a cellular immune response to haptens: the haptens were covalently coupled to mycobacteria and injected into animals in incomplete Freund's adjuvant. Humoral antibody was induced by coupling the same isomers to bovine serum albumin and injecting them into animals in complete Freund's adjuvant. Both groups of animals were examined for hapten-specific cellular immunity (migration inhibition factor, delayed skin tests) and for antibody response (passive hemolysin test). The results show that although both cell-mediated and humoral responses can discriminate the para, meta and ortho isomeric forms of the haptens used, the antibody response demonstrated much less cross-reactivity. The relevance of this finding to different requirement for B and T cells in antigenic recognition is discussed.

Existence of VHIII subgroup in rabbit antibody heavy chains.

Sequence analysis of CNBr fragments from the heavy chains of a rabbit anti-p-azobenzoate antibody of restricted heterogeneity has revealed a VHIII-like heavy chain sequence which was present in 6 to 10% of the heavy chain preparation.

Purification of DNP specific antibodies using sepharose bound DNP-para-aminobenzoylglutamate.

An immunoabsorbent column employing a DNP derivative having restricted molecular freedom (dinitrophenylated-para-aminobenzoylglutamate--Sepharose 4B) was prepared in order to isolate and purify DNP specific antibodies in high yield. The antibodies were recovered from the immunoabsorbent column in yields from 80 to 90% using 3 M sodium thiocyanate, and these antibodies retained their immunologic activity. There appeared to be limited selection of antibodies with specific affinities as was noted in a parallel experiment comparing a second antibody purification procedure.