Toxicity of an Fc-engineered anti-CD40 antibody is abrogated by intratumoral injection and results in durable antitumor immunity David A. Knorra,1, Rony Dahana,1,2, and Jeffrey V. Ravetcha,3 aLaboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, NY 10065 Contributed by Jeffrey V. Ravetch, August 16, 2018 (sent for review June 20, 2018; reviewed by Ronald Levy and Diane Mathis) Immune stimulation has emerged as a promising approach to the concentrations of antibody. Interestingly, while well tolerated at treatment of neoplastic diseases. Currently approved therapeutics, low doses up to 0.1 mg/kg, higher doses of anti-CD40 agonists such as anti-CTLA4 and anti-PD1, are primarily aimed at blocking led to profound transaminitis and hepatotoxicity. Compared with inhibitory signaling by immune cells. An alternative and poten- the parental antibody, 2141-V11 led to significantly higher tially synergistic approach would involve activation of immune transaminase levels [aspartate (AST) and alanine (ALT)] at pathways by agonism of stimulatory receptors, such as CD40. doses lower than the parent IgG2 antibody (Fig. 1A). When Agonistic antibodies, while promising in principle, have encoun- livers of treated mice were evaluated histologically, we found tered significant barriers in clinical trials limited by the systemic toxicity of such approaches. Using a mouse model humanized for evidence of both intravascular thrombi as well as hepatocyte both Fc receptors and CD40, we previously demonstrated en- necrosis in mice treated with 2141-V11 at concentrations equal hanced antitumor activity with an Fc-modified antibody. We now or above 0.25 mg/kg. No signs of hepatic toxicity were observed demonstrate that this model recapitulates the platelet and hepatic in histology evaluation of mice treated with 2141-V11 at toxicities seen with anti-CD40 antibodies in patients, providing a 0.125 mg/kg or lower doses, and in mice treated with the parental predictive measure of the dose-limiting activity of this approach. 2141-IgG2 at 0.2 mg/kg (Fig. 1B). The liver is a primary site of We further show that such toxicity can be circumvented and immune complex clearance in normal and pathologic conditions, INFLAMMATION durable systemic antitumor immunity achieved by intratumoral thus high levels of FcγRIIB on liver sinusoids (8), in addition to IMMUNOLOGY AND delivery of an Fc-engineered anti-CD40 agonistic antibody. activation of intrasinusoidal platelets (9–11), likely both con- tribute to this mechanism-based toxicity. Previous studies have CD40 | agonist antibody | immunotherapy | Fc receptor demonstrated that toxicity from a rat IgG2a CD40 antibody can be abrogated through pretreatment with anti–CSF1-R antibody he CD40 pathway provides a central mechanism for the ac- to deplete Kupffer cells (5), suggesting that FcγRIIB-expressing tivation of B cells, dendritic cells, and macrophages and is T Kupffer cells (12) within liver sinusoids may also play a direct well established as a powerful adjuvant in preclinical animal models. Despite its promise, clinical trials with agonistic, anti- role. Thus, not only does the 2141-V11 variant lead to enhanced CD40 antibodies have encountered dose-limiting toxicities and, as a consequence, minimal clinical responses (1). We engineered Significance the human anti-CD40 agonist antibody CP-870,893 (2–4) with five point mutations in the Fc domain selectively increasing its Antibodies blocking inhibitory checkpoints on T cells have been binding to human FcyRIIB (referred to here as “2141-V11”), a major advance in cancer treatment. However, agonistic an- and demonstrated that it has significantly enhanced antitumor tibodies have had less success due to toxicity concerns. Har- activity compared with its parental IgG2 version in several tumor nessing the knowledge that agonistic antibodies require the models (2). Using a mouse model carrying human Fcy receptors inhibitory Fc receptor (FcR), we engineered a CD40 antibody (FcyRs) and human CD40 (hFcyR/hCD40) in place of their with improved in vivo activity. Because current models fail to mouse homologs, we reported that, when given systemically, the recapitulate important dose-limiting toxicities in patients, we enhanced in vivo activity of the 2141-V11 was accompanied by developed a mouse model carrying human CD40 and FcRs. This increasing thrombocytopenia and transaminitis (2). These same model mirrors human toxicities and allowed for the develop- toxicities are seen with the current clinically used anti-CD40 ment of an in situ vaccination approach leading to durable tumor control. These results support the rational design of antibodies and the primary drivers of the dose-limiting toxic- immune modulating antibodies as well as stress the impor- ities, resulting from the expression of CD40 on platelets and tance, and possible reconsiderations needed, for optimal pre- their activation by agonistic anti-CD40 antibodies. In prepara- clinical models allowing parallel efficacy and toxicity analyses. tion for clinical studies of this Fc-engineered antibody we set out to optimize a dosing and delivery regimen that would result in Author contributions: D.A.K., R.D., and J.V.R. designed research; D.A.K. and R.D. per- minimal toxicity with optimal antitumor activity. formed research; D.A.K., R.D., and J.V.R. analyzed data; and D.A.K., R.D., and J.V.R. wrote the paper. Results Reviewers: R.L., Stanford University; and D.M., Harvard Medical School. The toxicity of rat IgG2a anti-CD40 antibodies has been pre- The authors declare no conflict of interest. viously established in mouse tumor models (5), with s.c. dosing Published under the PNAS license. allowing activity and better tolerability (6, 7). However, this has 1D.A.K. and R.D. contributed equally to this work. never been evaluated using human antibodies in a system 2Present address: Department of Immunology, Weizmann Institute of Science, Rehovot expressing both human CD40 and human FcRs. We previously 7610001, Israel. generated an Fc-enhanced anti-CD40 antibody (2141-V11) with 3To whom correspondence should be addressed. Email: [email protected]. superior in vivo activity to the currently available clinical re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. agents (2). To test the effects of 2141-V11 on liver function tests, 1073/pnas.1810566115/-/DCSupplemental. we treated hFcyR/hCD40 mice systemically with increasing www.pnas.org/cgi/doi/10.1073/pnas.1810566115 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 the MTD of 0.1 mg/kg would limit toxicity and allow antitumor A ALT AST 4000 6000 efficacy. When 2141-V11 and 2141-IgG2 were repeatedly ad- V11 V11 2000 3000 IgG2 IgG2 ministered as four consecutive doses (0.1 mg/kg and 0.2 mg/kg, 150 500 A L) L) / respectively) we did not observe any signs of toxicity (Fig. 2 ). / U 400 100 T(U Thus, when kept at their predetermined MTD, CD40 antibodies LT ( S 300 A A 200 50 are safe to be used in a multiple dose schedule. Next, using our 100 humanized model, MC38 tumors were allowed to engraft for 1 0 0 0.03125 0.125 0.5 2 0.03125 0.125 0.5 2 wk as in prior experiments, followed by four consecutive doses of Ab (mg/kg) Ab (mg/kg) 2141-V11, starting at day 7, given every 4 d. These studies B demonstrated that at their respective MTDs, 2141-V11 led to significantly better tumor control than the parental antibody with a human IgG2 Fc, 2141-IgG2 (Fig. 2B). These data also dem- onstrate that this approach limits toxicity as liver function tests and platelets remain similar to untreated controls at the end point of this experiment (Fig. 2C). However, as opposed to previous studies which used higher doses of the antibody (2), we did not achieve complete, durable tumor control with this ap- proach. These results suggest that when used in patients at their respective MTDs, increased therapeutic antitumor immunity can be achieved by the Fc-engineered 2141-V11 compared with its C parental IgG2 version; however, an optimal dose may never be reached for either subclass in humans when used at these doses. Because the goal of 2141-V11 treatment is to enhance acti- vation of intratumoral antigen-presenting cells, leading to the stimulation of cytotoxic T cells that may then migrate to distant tumor sites (referred to as an abscopal effect), we next tested whether or not direct delivery of 2141-V11 to the tumor site maintains antitumor activity with the additional benefit of po- tentially reducing the toxicities limiting optimal dosing in vivo (6, Fig. 1. Improved FcγRIIB binding enhances in vivo toxicity of anti-CD40 13, 14). Previous work has shown that the activity of 2141-V11 is antibodies. (A) Toxicity of liver transaminases in response to increasing lev- strictly dependent on its interactions with the inhibitory Fc re- els of anti-CD40 antibodies. Mice were treated with increasing doses of ceptor FcyRIIB; thus, we next assessed the relative percentages 2141-V11 or the parental IgG2 anti-CD40 antibody and liver transaminases of Fc receptors on leukocytes in the tumor microenvironment (AST and ALT) were measured. Data include three to five mice at each concentration. (B) Livers from mice treated with 2141-V11 show evidence of (TME). Although we previously demonstrated that humanized intravascular thrombi and hepatocyte necrosis. Mice treated with 2141-IgG2 mice phenocopy the CD40 and Fc receptor expression profile showed no toxicity at 0.2 mg/kg or below. Mice treated with 2141-V11 similar to that found in humans (2, 15), the relative levels of demonstrated evidence of intravascular thrombi and hepatocyte necrosis human FcyRs expressed within the TME has only been recently starting at dose 0.25 mg/kg or greater. Representative images from mice assessed in this model (16).