Antitumor Activity Of Antibodies Research Articles

The promising role of antibody drug conjugate in cancer therapy: Combining targeting ability with cytotoxicity effectively.

IntroductionTraditional cancer therapy has many disadvantages such as low selectivity and high toxicity of chemotherapy, as well as insufficient efficacy of targeted therapy. To enhance the cytotoxic effect and targeting ability, while reducing the toxicity of antitumor drugs, an antibody drug conjugate (ADC) was developed to deliver small molecular cytotoxic payloads directly to tumor cells by binding to specific antibodies via linkers.MethodBy reviewing published literature and the current progress of ADCs, we aimed to summarize the basic characteristics, clinical progress, and challenges of ADCs to provide a reference for clinical practice and further research.ResultsADC is a conjugate composed of three fundamental components, including monoclonal antibodies, cytotoxic payloads, and stable linkers. The mechanisms of ADC including the classical internalization pathway, antitumor activity of antibodies, bystander effect, and non‐internalizing mechanism. With the development of new drugs and advances in technology, various ADCs have achieved clinical efficacy. To date, nine ADCs have received US Food and Drug Administration (FDA) approval in the field of hematologic tumors and solid tumors, which have become routine clinical treatments.ConclusionADC has changed traditional treatment patterns for cancer patients, which enable the same treatment for pancreatic cancer patients and promote individualized precision treatment. Further exploration of indications could focus on early‐stage cancer patients and combined therapy settings. Besides, the mechanisms of drug resistance, manufacturing techniques, optimized treatment regimens, and appropriate patient selection remain the major topics.

Internalization by Cells and Antitumor Activity of Antibodies and Immunotoxins Specific for the Heat Shock Protein 90 β Isoform

Mouse monoclonal antibodies to Hsp90β (β isoform of heat shock protein 90) have been shown to bind specifically to Hsp90β localized on the surface of tumor and nontransformed cells. After binding to the membrane-associated Hsp90β, the antibodies actively dissociated into the culture medium and were also internalized by the cells. An immunoconjugate based on the Hsp90β-specific antibody and the cytotoxic agent mertansine did not have high cytotoxic activity for tumor cells in vitro. Administration of Hsp90β-specific antibodies in mice did not affect the growth of the primary Lewis lung carcinoma, while tumor metastasis to the lungs decreased and the average lifespan of mice increased. The results indicate a certain therapeutic potential of antibodies to Hsp90β for the treatment of tumor diseases.

Abstract 4110: Identification of PD1 B cell mimotopes with functional PD1-PDL1 blocking capacity: New strategy for cancer immunotherapy

Abstract Immune checkpoint (ICP) blockade has become a major focus in cancer immunotherapy, and the application of monoclonal antibodies (mAbs) as ICP inhibitors shows impressive results in the therapy against cancers like melanoma, renal cell carcinoma or non-small cell lung cancer. Active immunization with B cell epitopes/mimotopes of ICP inhibitors rather than application of the corresponding mAbs for passive immunotherapy may, however, provide advantages such as induction of antibodies by the patient’s own immune system and overcoming the costly treatment of the mAbs. Applying a platform for bacterial surface-display of overlapping peptides spanning the extracellular domains of human PD1 (hPD1) and mouse PD1 (mPD1), as well as in vitro solid phase-based ELISA assay and cellular-based assays involving human Jurkat T cells expressing human/mouse PD1 and/or PDL1, B cell epitopes/mimotopes of Nivolumab and an anti-mPD1 mAb (with a blocking capacity) were identified. The mimotopes were not only shown to inhibit the binding of the corresponding mAbs but also to revert the PD1-PDL1 blocking inhibitory capacity of the respective mAbs. More importantly, to proof the antitumor activity of antibodies against PD1 mimotopes in vivo, rabbits were immunized with the mimotope of anti-mPD1 mAb to generate mimotope-specific IgGs. The specific IgGs not only inhibited the interaction of mPD1 and mPDL1 in vitro, but also significantly reduced tumor growth in a syngeneic model of mice grafted with cells expressing the breast cancer-associated tumor antigen Her-2/neu. Our results show systematic identification and characterization of mimotopes of anti-PD1 ICP inhibitor B cell mimotopes conferring in vivo anti-tumor activity, and also indicate the potential of such mimotopes for active immunization in cancer therapy. Based on the results, enhancement of our second generation of anti-Her-2/neu vaccine (HerVAXX; Tobias et al, 2017*) by combining with mimotopes of ICP inhibitors is also proposed, which may potentially pave the way to a paradigm change in active immunotherapy against Her-2/neu overexpressing cancers using an effective multi-level vaccine. The study is granted by Imugene Limited, Australia, and Medical University of Vienna. * Tobias J. et al. BMC Cancer, 2017, 17:118. Citation Format: Joshua Tobias, Claire Battin, Annika De Sousa Linhares, Baier Karin, Katharina AMbroye, Mirjana Drinic, Erika Garner-Spitzer, Christoph Zielinski, Michael Kundi, Peter Steinberger, Ursula Wiedermann. Identification of PD1 B cell mimotopes with functional PD1-PDL1 blocking capacity: New strategy for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4110.

Antitumor activity of antibody against cytotoxic T lymphocyte epitope peptide of lymphocyte-specific protein tyrosine kinase.

Although humoral responses against CTL epitope peptides from lymphocyte‐specific protein tyrosine kinase (Lck) antigen have been observed in the majority of healthy donors and cancer patients, the biological activity of the antibody has not been determined. We investigated the biological activity of mAb against CTL epitope peptide of Lck antigen at positions 486‐494 (anti‐Lck‐486 mAb). This mAb induced dendritic cell maturation from murine bone marrow cells by the immune complex form in vitro, and inhibited tumor growth in association with a suppression of tumor‐infiltrating T cells, including T regulatory cells in a murine model using female BALB/cCrlCrlj mice (H‐2Kd). More potent tumor inhibition was observed when this mAb was given prior to peptide vaccination. These results may help to unveil the biological activity of anti‐Lck peptide antibodies against CTL epitope peptides.

Abstract 2319: Selinexor, a selective inhibitor of nuclear export (SINE), enhances the in vivo efficacy of checkpoint blockade with antibodies targeting CTLA4 or PD-1/PD-L1 in melanoma

Abstract Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound that has been administered to >1000 cancer patients in Phase I and II trials to date, with evidence of efficacy and tolerability. This small molecule targets exportin-1 (XPO1), a key nuclear export protein with >200 cargo proteins which include both tumor suppressors and cell cycle modulators. As a result, selinexor blocks nuclear export of proteins including IκB, NFAT1c, STAT1 and STAT3, which regulate expression of the inhibitory T cell receptors CTLA4, PD1 and its ligand, PD-L1. We hypothesized that selinexor would upregulate T cell checkpoint molecule expression, and thereby enhance the anti-tumor activity of antibodies targeting PD-1/PD-L1 or CTLA4. Human (A375, CHL-1) and murine (B16F10) melanoma cell lines expressed high levels of PD-L1 protein at baseline, and PD-L1 expression was induced following selinexor treatment in numerous other tumor cell lines (including HCT-116, MDA-MB-468, MV-4-11, OVCAR-8, and PC-3). Examination of lymphocytes revealed that selinexor also increased expression of PD-1 and CTLA4 by ∼2-fold. Mice bearing syngeneic B16F10 melanoma tumors treated with selinexor (15 mg/kg 2 x weekly) and anti-CTLA4 (250 μg, 2 x weekly) demonstrated a significant reduction in tumor growth rate (p = 0.0065) while monotherapy had no significant effect on tumor growth. Similar results were obtained in mice bearing B16F10 melanoma treated with the combination of selinexor + anti-PD-1 (200 μg, 2 x weekly, p < 0.034) or selinexor + anti-PD-L1 (100-200 μg, 2 x weekly, p < 0.001). Importantly, no weight loss or signs of toxicity were evident in any in vivo study. Further immunophenotypic analyses have been completed in animals receiving selinexor alone or in combination with anti-PD-L1. In combination treated mice, we observed a significantly increased percentage of splenic NK cells (p ≤ 0.050), and a significantly increased percentage of splenic Th1 T cells (p≤0.011), all compared to vehicle treated mice. Interestingly, combining selinexor with anti-PD-L1 significantly decreased the percentage of splenocytes that expressed PD-L1 (p<0.001). These changes are indicative of increased anti-tumor immune activity; however, they were accompanied by significantly increased percentages of myeloid cell subsets in combination treated mice (p ≤ 0.050). The immunologic significance of this myeloid cell expansion is currently under investigation. These data indicate that the efficacy of selinexor may be enhanced by disrupting immune checkpoints in effector cells (T and NK cells). This provides data in support of novel, evidenced-based combinations involving immunotherapy with XPO1 inhibition that deserve further investigation for advanced cancer. Citation Format: Matthew R. Farren, Reena Shakya, Rebecca Hennessey, Thomas Mace, Jennifer Yang, Omar Elnaggar, Gregory Young, Yosef Landesman, Robert Carlson, Sivan Elloul, Marsha Crochiere, Christin Burd, Gregory B. Lesinski. Selinexor, a selective inhibitor of nuclear export (SINE), enhances the in vivo efficacy of checkpoint blockade with antibodies targeting CTLA4 or PD-1/PD-L1 in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2319.

Abstract 660: Engineering strategies to control stoichiometry, site of conjugation, serum stability and effector functions of antibody-drug conjugates

Abstract A promising approach to enhance the antitumor activity of antibodies and reduce the systemic toxicity of small molecule anti-cancer drugs is to covalently attach the small molecule to the antibody thus forming antibody drug conjugates (ADCs). Conventionally, ADCs are produced by chemical conjugation of drugs to antibodies through either lysine side chain amines or native cysteine sulfhydryl groups generated by reduction of interchain disulfide bonds. Both of these methods yield heterogeneous conjugates with complex biophysical properties. To limit these liabilities, we have designed, characterized, and validated antibody variants, which allow precise control of the site of conjugation and the drug load; such as two and/or four drugs per antibody can be site-specific conjugated. These engineered antibody variants can be efficiently and site-specifically conjugated with drug payloads at milligram and gram scales to yield homogenous ADC products. Engineered antibody variants described here retain antigen-binding affinities, FcRn binding, and have biochemical, biophysical and manufacturability properties similar to their non-engineered parent antibodies. Upon conjugation to highly cytotoxic drugs with anti-mitotic and/or DNA alkylating activities, the antibody variants have potent in vitro and in vivo anti-tumor activity. Furthermore, we describe ADCs vehicles that provide efficient scaffolds for site-specific conjugation of drugs while offering the opportunity to minimize off-target toxicities often observed with ADCs as a result of their binding to cells expressing Fc-gamma receptors. Finally, we show the impact of the identified sites for drug conjugation on the in vivo pharmacokinetic and safety profile of ADCs in rats. Our strategies for engineering ADCs with defined sites for conjugation, controlled drug loads, potentially decreased off-target toxicity, and optimal serum stability are broadly applicable to any full-length IgG or Fc-containing therapeutics as they involve the Fc constant domain of the antibody. Citation Format: Nazzareno Dimasi, Ryan Fleming. Engineering strategies to control stoichiometry, site of conjugation, serum stability and effector functions of antibody-drug conjugates. [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 660. doi:10.1158/1538-7445.AM2014-660

Study of Antitumor Activity of Antibodies to TNF-α on Walker Carcinosarcoma Model

The effect of release-active antibodies to TNF-α (Artrofoon) on the development of Walker carcinosarcoma 256 was studied in Wistar rats. Intragastric dose of this drug significantly inhibited the growth of tumor node (55% inhibition of tumor growth), but less effectively than the reference drug cyclophosphamide (80%). The studied drug significantly prolonged animal' lifespan (+97%) and its efficiency in this respect was comparable to that of cyclophosphamide (+96%).

Role of the immunoglobulin constant region in the antitumor activity of antibodies to cytotoxic T-lymphocyte antigen-4 (CTLA-4).

9055 Background: Anti-CTLA-4 therapy enhances antitumor T-cell responses by both cell-intrinsic and cell-extrinsic mechanisms. Effector T-cell (Teff) activation is increased directly by interfering with CTLA-4-B7 interactions that negatively regulate T cells and by interfering with CTLA-4 expressed on regulatory T cells (Tregs), which function to inhibit immune responses. To analyze in greater detail the mechanism of action of anti-CTLA-4 antibodies (Abs), we examined the role of the immunoglobulin constant region in the antitumor activity of anti-CTLA-4 Abs in mouse tumor models. Methods: The activity of anti-CTLA-4 Abs with different mouse IgG constant regions were compared in several mouse tumor models, and intratumoral and peripheral T cells were analyzed by FACS. DNA samples from 488 patients with metastatic melanoma who received ipilimumab in a phase III clinical trial, MDX010-20, were analyzed for polymorphisms in the IgG fragment c receptor (FcR) at FCGR3A (V158F) and FCGR2A (H131R) loci. Results: In subcutaneous MC38 and CT26 colon tumor models, an anti-CTLA-4 Ab containing the mouse IgG2a constant region exhibited enhanced antitumor activity compared to an anti-CTLA-4 Ab containing the IgG2b constant region, while anti-CTLA-4 Abs containing mouse IgG1 or a mutated mouse IgG1 D265A constant region showed no activity. Anti-CTLA-4-IgG2a caused a dramatic reduction of Tregs at the tumor site that resulted in a greater Teff/Treg ratio. In contrast, all isotypes resulted in expansion of Tregs in the periphery. These results point to an important role for FcR in the action of anti-CTLA-4 Abs. In patients from study MDX010-20, there was no association between the 2 FcR polymorphisms (FCGR3A and FCGR2A) and overall survival. Conclusions: These preclinical studies reveal a novel dual activity of anti-CTLA-4 Abs consisting of intratumoral reduction of Tregs together with activation of Teff cells. This effect is mediated by the constant region and is presumably due to antibody-dependent cellular cytotoxicity. The data suggest that FcR-bearing cells at the tumor site, as well as the presence of intratumoral Tregs, may be important factors in the antitumor activity of anti-CTLA-4 Abs.

Apoptotic and antitumor activity of death receptor antibodies require inhibitory Fcγ receptor engagement

By virtue of their ability to induce apoptosis and regulate growth, differentiation, and cytokine responses, the tumor necrosis factor receptor (TNFR) superfamily members have emerged as attractive targets for anticancer therapeutics. Agonistic antibodies to apoptosis-inducing TNFRs, such as death receptor 5 (DR5), although displaying impressive activities against a variety of tumors in preclinical models, appear to be less active in clinical trials. We report that the in vivo apoptotic and antitumor activities of these antibodies have an absolute requirement for the coengagement of an inhibitory Fcγ receptor, FcγRIIB. Anti-DR5 antibodies of the type currently in clinical trials have weak FcγRIIB binding and thus are compromised in their proapoptotic and antitumor activities in both colon and breast carcinoma models. Enhancing FcγRIIB engagement increases apoptotic and antitumor potency. Our results demonstrate that Fc domain interactions are critical to the therapeutic activity of anti-DR5 antibodies and, together with previous reports on agonistic anti-CD40 antibodies, establish a common requirement for FcγRIIB coengagement for optimal biological effects of agonistic anti-TNFR antibodies.

A phase II study of the anti-CTLA4 monoclonal antibody (mAb), CP-675,206, in patients with refractory metastatic adenocarcinoma of the colon or rectum

3035 Background: The antitumor activity of antibodies to cytotoxic T lymphocyte-associated antigen 4 (CTLA4) has been demonstrated in a variety of murine tumor models, including rejection of established tumors and secondary exposure to tumor cells. This suggests that blockade of the inhibitory effects of CTLA4 can promote effective antitumor immune responses. CP-675,206 has also been shown to induce durable tumor responses in patients (pts) with metastatic melanoma in phase 1 and phase 2 clinical studies. The purpose of this study was to assess safety and efficacy of CTLA4 blockade with the fully human mAb CP-675,206 as single-agent therapy in pts with relapsed/ refractory colorectal cancer. Methods: A single-arm, multicenter, phase II trial of CP-675,206 was conducted in pts with measurable adenocarcinoma of the colon or rectum failing standard treatments and with an ECOG performance status of 0 or 1. Patients received 15 mg/kg Q90 days via IV infusion until disease progression. The primary objective was response rate by RECIST criteria. Secondary objectives included safety, duration of response, progression-free survival, and overall survival. Results: A total of 47 pts who received a median of 4 previous therapies (range, 1 to 9) were treated, and 46 experienced disease progression or death because of disease before reaching the planned second dose at 3 months. Grade 3 or 4 adverse events attributed to study drug were limited to diarrhea (n = 3, 6.4%) and idiopathic thrombocytopenia purpura (n = 1, 2.1%). Four pts (8.5%) had grade 2 diarrhea. Four pts received steroids and 2 received infliximab. One patient was removed for toxicity (diarrhea in the setting of what appeared to be treatment-related ulcerative colitis that was responsive to steroids). One patient (2%; 95% CI = 0%, 11%) had a stable ovarian mass and a substantial regression in an adrenal mass. This patient is continuing on study and has received a second dose. Conclusions: In heavily pretreated pts with colorectal cancer and good performance status, CP- 675,206 was tolerable. However, in this setting, CP-675,206 at 15 mg/kg did not demonstrate substantial single-agent activity. No significant financial relationships to disclose.

Inhibition of tumor growth by poly(ethylene glycol) derivatives of anti-ErbB2 antibodies.

Poly(ethylene glycol) (PEG) modification of substances with antitumor activity was shown to enhance penetration into growing solid tumors and extend antitumor effects. Accordingly, PEG was introduced as a modifier to two types of monoclonal antibodies (N12 and L26) specific to the ErbB2 (HER2) oncoprotein. These antibodies suppress the growth of tumors overexpressing ErbB2 (e.g. N87 human tumor) and the effect of PEG on their antitumor activity was evaluated. Methoxy-PEG-maleimide conjugated to sulfhydryl groups at the hinge region of the antibodies impaired their antibody binding to N87 tumor cells and did not enhance the antitumor inhibitory activity in tumor-bearing mice. A branched N-hydroxysuccinimide-activated PEG (PEG2), conjugated through amino groups of the protein, was used for binding to the whole antibody (Ab) or to its monomeric Fab' fragment. When tested against N87 cells in vitro, the binding activity and antitumor cytotoxic effects of Ab-PEG2 were mostly preserved. PEG2 modification did not seem to alter the tumor-inhibitory activity of the antibodies in vivo and the same pattern of tumor development was observed during the first few weeks following administration. However, the stimulating effects of PEG were observed at later stages of tumor growth since tumor development was either slowed down or completely arrested. Furthermore, a second tumor implanted into the same mice during this later stage was significantly or completely inhibited, as compared to results in mice injected with the unmodified antibody. The Fab'-PEG2 monomeric derivative was also shown to be effective in inhibiting the growth of a second tumor. The extended and prolonged enhancing effect of PEG on the antitumor activity of antibodies or Fab' fragments directed against ErbB2 may be of importance in the treatment of ErbB2-overexpressing neoplasms.