SUMMARY Adaptation of the endoplasmic reticulum (ER) pathway for MHC class I (MHC-I) presentation in dendritic cells enables cross-presentation of peptides derived from phagocytosed microbes, infected cells, or tumor cells to CD8 T cells. How these peptides intersect with MHC-I molecules remains poorly understood. Here, we show that MHC-I selectively accumulate within phagosomes carrying microbial components, which engage Toll-like receptor (TLR) signaling. Although cross-presentation requires Sec22b-mediated phagosomal recruitment of the peptide loading complex from the ER-Golgi intermediate compartment (ERGIC), this step is independent of TLR signaling and does not deliver MHC-I. Instead, MHC-I are recruited from an endosomal recycling compartment (ERC), which is marked by Rab11a, VAMP3/cellubrevin, and VAMP8/endobrevin and holds large reserves of MHC-I. While Rab11a activity stocks ERC stores with MHC-I, MyD88-dependent TLR signals drive IκB-kinase (IKK)2-mediated phosphorylation of phagosome-associated SNAP23. Phospho-SNAP23 stabilizes SNARE complexes orchestrating ERC-phagosome fusion, enrichment of phagosomes with ERC-derived MHC-I, and subsequent cross-presentation during infection.

Cross-presentation involves the presentation of peptides derived from internalized cargo on major histocompatibility complex class I molecules by dendritic cells, a process critical for tolerance and immunity. Detailed studies of the pathways mediating cross-presentation have revealed that this process takes place in a specialized subcellular compartment with a unique set of proteins. In this review, we focus on the recently appreciated role for intracellular vesicular traffic, which serves to equip compartments such as endosomes and phagosomes with the necessary apparatus for conducting the various steps of cross-presentation. We also consider how these pathways may integrate with inflammatory signals particularly from pattern recognition receptors that detect the presence of microbial components during infection. We discuss the consequences of such signals on initiating cross-presentation to stimulate adaptive CD8 T cell responses.

CD33 is expressed in 90% of patients with acute myeloid leukemia (AML), and its extracellular portion consists of a V domain and a C2 domain. A recent study showed that a single nucleotide polymorphism (SNP), rs12459419 (C > T), results in the reduced expression of V domain–containing CD33 and limited efficacy of V domain–binding anti-CD33 antibodies. We developed JNJ-67571244, a novel human bispecific antibody capable of binding to the C2 domain of CD33 and to CD3, to induce T-cell recruitment and CD33+ tumor cell cytotoxicity independently of their SNP genotype status. JNJ-67571244 specifically binds to CD33-expressing target cells and induces cytotoxicity of CD33+ AML cell lines in vitro along with T-cell activation and cytokine release. JNJ-67571244 also exhibited statistically significant antitumor activity in vivo in established disseminated and subcutaneous mouse models of human AML. Furthermore, this antibody depletes CD33+ blasts in AML patient blood samples with concurrent T-cell activation. JNJ-67571244 also cross-reacts with cynomolgus monkey CD33 and CD3, and dosing of JNJ-67571244 in cynomolgus monkeys resulted in T-cell activation, transient cytokine release, and sustained reduction in CD33+ leukocyte populations. JNJ-67571244 was well tolerated in cynomolgus monkeys up to 30 mg/kg. Lastly, JNJ-67571244 mediated efficient cytotoxicity of cell lines and primary samples regardless of their SNP genotype status, suggesting a potential therapeutic benefit over other V-binding antibodies. JNJ-67571244 is currently in phase 1 clinical trials in patients with relapsed/refractory AML and high-risk myelodysplastic syndrome.

Redirecting T cells to specifically kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of blinatumomab for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. In hematological malignancies, the bone marrow (BM) niche is protective to leukemic stem cells and has minimized the efficacy of several anti-cancer drugs. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using bispecific antibodies targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia or multiple myeloma cells with BM stromal cells protected tumor cells from bispecific antibody-T cell-mediated lysis in vitro and in vivo. Impaired CD3 redirection cytotoxicity was correlated with reduced T cell effector responses and cell-cell contact with stromal cells was implicated in reducing T cell activation and conferring protection of cancer cells. Finally, blocking the VLA4 adhesion pathway in combination with CD3 redirection reduced the stromal-mediated inhibition of cytotoxicity and T cell activation. Our results lend support to inhibiting VLA4 interactions along with administering CD3 redirection therapeutics as a novel combinatorial regimen for robust anticancer responses.

Classic MHC-I presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8 + T cells. Priming CD8 + T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells (DCs). We found that protective CD8 + T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment (ERC) of MHC-I and as such impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I accumulated in ER-Golgi intermediate compartments (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and ERC-dependent cross-presentation are impaired in DCs, cell-autonomous non-canonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8 + T cell priming.

CD33 is expressed in 90% of acute myeloid leukemia (AML) patients with expression in both blasts and leukemic stem cells. The extracellular portion of CD33 consists of a V and a C2 Ig-like domain. A recent study showed that a single nucleotide polymorphism (SNP) rs12459419 (C >T; Ala14Val in exon 2 of CD33) was present in ~50% of the Western AML population and is associated with preferential expression of an alternatively spliced CD33 isoform lacking exon2, resulting in the deletion of the CD33 V domain. Interestingly, several CD33-antibody-based therapies, including gemtuzumab ozogamicin (GO), the only approved anti-CD33 antibody drug conjugate for AML, bind and recognize the V domain of CD33. Recent data demonstrated that patients with SNP rs12459419 CC genotype receiving GO had a significantly lower risk of relapse and increased event-free survival compared to patients with the CT or TT genotypes. Given the data with GO, it is reasonable to hypothesize that the activity of other V binding CD33 antibodies maybe limited to a subset (~50%, rs12459419 CC genotype) of AML patients. On the other hand, since the C2 domain is shared by all CD33 isoforms in AML patients, we hypothesized that a C2 binding anti-CD33 antibody could target AML cells more broadly regardless of their SNP status. Additionally, we reasoned that targeting the membrane proximal C2 domain would be a beneficial strategy for a CD3 redirection bispecific antibody as targeting epitopes closer to the membrane have been reported to mediate efficient synapse formation between T cells and target cells leading to potent anti-tumor responses. We developed JNJ-67571244, a novel human bispecific antibody capable of binding to the C2 domain of CD33, and to CD3 to induce T-cell recruitment and tumor cell cytotoxicity independently of their SNP status. JNJ-67571244 specifically binds to CD33-expressing target cells and induces cytotoxicity of CD33+ AML cell lines in vitro at 48 hours (EC50 values: KG-1=0.168 nM, MOLM-13=0.131 nM, Kasumi-1=0.05 nM and OCI-AML3=0.183 nM) with concomitant T cell activation (EC50 values: KG-1=0.066 nM, MOLM-13=0.028 nM, Kasumi-1=0.043 nM and OCI-AML3=0.05 nM) along with cytokine release. In contrast, JNJ-67571244 was unable to kill CD33- cancer cell lines (CARNAVAL and KG-1 cells with a genetic deletion of CD33), demonstrating the specificity of the cytotoxicity. JNJ-67571244 demonstrated statistically significant anti-tumor activity in vivo in established disseminated and subcutaneous mouse models of human AML (MOLM-13Luc and KG-1: up to 100% and 92% tumor growth inhibition respectively) through T cell redirection activity. Furthermore, this antibody could deplete CD33+ blasts (EC50=0.549 nM) in AML patient blood samples (n=7) in an ex-vivo assay at 48 hours with concurrent T cell activation (EC50=0.355 nM). JNJ-67571244 also cross-reacts with cyno CD33 and CD3 and was well-tolerated in cynomolgus monkeys up to 30 mg/kg along with a sustained reduction in CD33+ leukocyte populations. Lastly, JNJ-67571244 mediated efficient cytotoxicity of cell lines and primary samples regardless of their genotype status (SNP rs12459419 CC, CT and TT), suggesting a potential therapeutic advantage over competitor V-binding antibodies. JNJ-67571244 is currently in Phase 1 clinical trials to treat relapsed/refractory AML and high risk myelodysplastic syndrome (MDS) patients (NCT03915379). Citation Format: Priyanka Nair-Gupta, Michael Diem, Dara Reeves, Weirong Wang, Robert Schulingkamp, Katrin Sproesser, Bethany Mattson, Bradley Heidrich, Jocelin Joseph, Jocelyn Sendecki, Brad Foulk, Gerald Chu, Damien Fink, Qun Jiao, Sheng-Jiun Wu, Kathryn Packman, Yusri Elsayed, Ricardo Attar, Francois Gaudet. JNJ-67571244: A novel anti-CD33 C2 domain binding bispecific antibody with potent T cell redirection activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5662.

Redirecting T cells to specifically target and kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of CD19xCD3 BiTE (Blincyto) for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. For instance, the bone marrow (BM) niche is appreciated to be a site of immune privilege at steady state to allow for normal hematopoiesis and immune cell generation. Additionally, in hematological malignancies, the BM niche is protective to leukemic stem cells, a phenomenon that has minimized the efficacy of several anti-cancer drugs including chemotherapy, targeted small molecule inhibitors and antibody based therapies. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using antibodies made with the Genmab DuoBody® technology targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia (AML; KG-1, MOLM-13 and OCI-AML5) or multiple myeloma (MM; H929 and RPMI8226) cell lines with bone marrow stromal cell lines (HS-5 and HS-27a) significantly protected leukemic cells from DuoBody®-T cell mediated lysis in vitro. Specifically, co-culture of bone marrow stromal cells in killing assays led to a 20-50% decrease in the maximum observed cytotoxicity and a 3-10 fold weaker EC50, reflecting an impact on both the efficacy and potency of bispecific DuoBody® antibodies. Similar results were also observed with primary stromal cells obtained from healthy donors. Furthermore, presence of stromal cells in a humanized xenograft AML model attenuated tumor growth inhibition (TGI) observed with DuoBody® treatment (78% TGI with MOLM-13 vs 15% TGI with MOLM-13+HS-5). Impaired TGI correlated with reduced T cell activation (7 fold decrease in CD25 upregulation) and production of granzyme B (8 fold reduction), providing one potential mechanism to explain loss of activity of the DuoBody® antibody. In vitro trans-well redirection assays revealed that cell-cell contact with stromal cells was crucial for reduced T cell activation and target cell survival relative to controls. Additionally, leukemic cells not killed by T cells were observed to preferentially cluster around stromal cells. We propose that target cells can evade T cell death by a stromal cell dependent mechanism involving activation of multiple pro-survival and anti-apoptotic pathways in leukemic cells in addition to suppressed activation of T cells. We are currently studying pathways mediating the cross-talk between cancer, immune and stromal cells. A better understanding of the mechanisms underlying the protective and immunosuppressive nature of the BM microenvironment will be instrumental to the design of more effective CD3 redirected therapeutics or novel combinatorial regimens for robust anti-cancer responses. Disclosures Nair-Gupta: Janssen: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Smith:Janssen Research & Development, LLC: Employment, Equity Ownership. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Pillarisetti:Janssen Research and Development, LLC: Employment. Baldwin:Janssen: Employment. Packman:Janssen: Employment. Elsayed:Janssen: Employment. Attar:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued.