Autolus (United Kingdom)

6576 Background: CD19 chimeric antigen receptor T-cell therapy (CAR T) exhibits good efficacy in adults with R/R B-ALL but is associated with higher toxicity with increasing age. Obe-cel, an autologous anti-CD19 CAR T, has shown high and durable response rates with low incidence of immunotoxicity in adult R/R B-ALL, and was recently approved by the US FDA. Here, we report a post-hoc analysis of the Phase Ib/II FELIX trial (NCT04404660) evaluating efficacy, safety, and persistence outcomes with obe-cel stratified by pt age. Methods: Adult R/R B-ALL pts received obe-cel using a tumor burden-guided dosing strategy to minimize toxicity. Overall remission rate (ORR; complete remission [CR]/CR with incomplete hematologic recovery), event-free survival (EFS), safety, and persistence are reported for pts aged <55 and ≥55 years (yrs; data cut-off: 7 Feb 2024). Results: Of 127 obe-cel infused pts, 79 (62.2%) were aged <55 yrs (median 36.0 [range: 20–54]) and 48 (37.8%) were aged ≥55 yrs (median 65.0 [range: 55–81]). A higher proportion of pts aged <55 yrs were Hispanic/Latino (36.7% vs 18.8%), had extramedullary disease at lymphodepletion (LD; 29.1% vs 8.3%), received prior blinatumomab (53.2% vs 22.9%), and prior inotuzumab ozogamicin (35.4% vs 25.0%) than those ≥55 yrs, while a higher proportion of pts aged ≥55 yrs had Philadelphia chromosome-positive disease (47.9% vs 16.5%). Median bone marrow blast burden at LD was higher in pts aged ≥55 yrs (45.5%) vs <55 yrs (30.0%). At 21.5 months’ (mos) median follow-up (range: 8.6–41.4), the ORR (95% CI) was 72.2% (60.9–81.7) in pts aged <55 yrs vs 87.5% (74.8–95.3) in pts aged ≥55 yrs. In responders, 84.2% of pts <55 yrs and 83.3% ≥55 yrs with ≥1 post-infusion next-generation sequencing result achieved measurable residual disease-negative remission to 10 –6 leukemic cells by Month 3. Durable remission at 1 yr post infusion was observed in 68.3% and 51.8% of pts aged <55 and ≥55 yrs, respectively. EFS was comparable in pts aged <55 and ≥55 yrs: median (95% CI) 14.3 mos (6.0–not estimable [NE]) vs 11.7 mos (6.6–NE), respectively. While in remission, 29.8% of pts aged <55 yrs and 2.4% aged ≥55 yrs proceeded to consolidative stem cell transplant (SCT). Incidence of Grade ≥3 cytokine release syndrome (CRS; 2.5% vs 2.1%) and immune effector cell-associated neurotoxicity syndrome (ICANS; 5.1% vs 10.4%) were low for pts aged <55 and ≥55 yrs, respectively. Treatment-related mortality within 3 mos post obe-cel infusion was 0% in pts aged <55 yrs vs 4.2% in pts aged ≥55 yrs. CAR T-cell persistence was similar in both age groups. Conclusions: Obe-cel treatment resulted in favorable ORR and EFS with low Grade ≥3 CRS/ICANS incidence in both age groups. These findings indicate that obe-cel is effective and has a positive benefit/risk profile regardless of age, including in older adults with R/R B-ALL despite few receiving consolidative SCT. Clinical trial information: NCT04404660 .

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<div>Abstract<p>Adoptive T-cell therapy aims to achieve lasting tumor clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T-cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilizing non-native dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilizing the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimicked the cytokine receptor heterodimerization, with transcriptomic signatures like those obtained by activation of the native IL2 receptor. Moreover, we found that this dimerization structure was agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs co-expressed with a clinically relevant solid tumor–specific GD2-specific CAR. Based on this characterization, we suggest that the co-expression of either the common β-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T-cell expansion, engraftment, and efficacy. Our results demonstrate how Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T-cell therapies, as well as therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T-cell therapies.</p></div>

Adoptive T-cell therapy aims to achieve lasting tumor clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T-cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilizing nonnative dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilizing the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimicked the cytokine receptor heterodimerization, with transcriptomic signatures like those obtained by activation of the native IL2 receptor. Moreover, we found that this dimerization structure was agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs coexpressed with a clinically relevant solid tumor-specific GD2-specific chimeric antigen receptor (CAR). Based on this characterization, we suggest that the coexpression of either the common β-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T-cell expansion, engraftment, and efficacy. Our results demonstrate how Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T-cell therapies, as well as therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T-cell therapies.

BackgroundWe used a proliferating ligand (APRIL) to construct a ligand-based third generation chimeric antigen receptor (CAR) able to target two myeloma antigens, B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor.MethodsThe APRIL CAR was evaluated in a Phase 1 clinical trial (NCT03287804, AUTO2) in patients with relapsed, refractory multiple myeloma. Eleven patients received 13 doses, the first 15×106 CARs, and subsequent patients received 75,225,600 and 900×106 CARs in a 3+3 escalation design.ResultsThe APRIL CAR was well tolerated. Five (45.5%) patients developed Grade 1 cytokine release syndrome and there was no neurotoxicity. However, responses were only observed in 45.5% patients (1×very good partial response, 3×partial response, 1×minimal response). Exploring the mechanistic basis for poor responses, we then compared the APRIL CAR to two other BCMA CARs in a series of in vitro assays, observing reduced interleukin-2 secretion and lack of sustained tumor control by APRIL CAR regardless of transduction method or co-stimulatory domain. There was also impaired interferon signaling of APRIL CAR and no evidence of autoactivation. Thus focusing on APRIL itself, we confirmed similar affinity to BCMA and protein stability in comparison to BCMA CAR binders but reduced binding by cell-expressed APRIL to soluble BCMA and reduced avidity to tumor cells. This indicated either suboptimal folding or stability of membrane-bound APRIL attenuating CAR activation.ConclusionsThe APRIL CAR was well tolerated, but the clinical responses observed in AUTO2 were disappointing. Subsequently, when comparing the APRIL CAR to other BCMA CARs, we observed in vitro functional deficiencies due to reduced target binding by cell-expressed ligand.

ABSTRACTThe human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of theCoronaviridaefamily SARS-CoV-1 and HCoV-NL63. Here we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7 and B.1.351 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralising antibody approaches. Furthermore, we report a pre-clinical characterisation package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralisation capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralised four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections.

Abstract Background: Mature T cell lymphomas are aggressive, treatment resistant cancers that are associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy T cells. Unlike B cell depletion, pan T cell aplasia is prohibitively toxic. The mutually exclusive expression of TCR β chains (TRBC) 1 or 2 allows targeting the malignant T cell population while preserving T cell function. Notably, a two amino acid inversion is the only exposed and antibody-accessible feature differentiating the two isoforms, making the development of therapeutics targeting TRBC1 and TRBC2 challenging. We have previously described a TRBC1 specific antibody which has been incorporated into a Chimeric Antigen Receptor (CAR) (Maciocia et al, Nat Med, 2017). Here we describe the derivation and characterization of a TRBC2 specific binder and CAR. In addition, we investigate use of these selective binders as Antibody Drug Conjugates (ADCs) for the treatment of HTLV-1 associated leukemia/lymphoma for which CAR T cell therapy may not be well suited. Methods: Anti TRBC2 antibodies were derived through crystallography and structural engineering of the previously described TRBC1 specific antibody. Briefly, TRBC2 binders were obtained by screening of small libraries of the TRBC1 binder with randomizations in key residues identified by crystallographic data and in silico design. TRBC2 binder candidates were first optimized for CAR function. Subsequently, TRBC2 specific CAR T cells were evaluated in vitro and in vivo for anti-tumor activity and selectivity. TRBC1/TRBC2 targeting antibodies, were further characterized as ADCs for biophysical properties, antibody internalization and cytotoxic function. Results: In vitro testing of TRBC2 CARs showed comparable efficacy to the previously described TRBC1 CAR. TRBC2 CAR T cells were effective at killing TRBC2 target cells while sparing TRBC1 positive cells. In vivo mouse models demonstrated that both TRBC1 and TRBC2 directed CARs could target their respective antigens with a high degree of specificity. TRBC1 and TRBC2 specific antibodies were used to generate ADC molecules as a proof of concept. Anti-TRBC1/TRBC2 antibodies were internalized and showed potential as ADCs. Interestingly, we demonstrate that an optimal affinity window facilitates improved antibody uptake and have further engineered both our TRBC1 and TRBC2 antibodies to take advantage of this particularity. Conclusions: Following on from structural and library-based approaches to generate CAR T cells capable of specifically targeting TRBC2, we have further characterized TRBC2 specific CAR T cells in vitro and in vivo. We have shown that highly specific antibodies, engineered as targeting moieties for TRBC1 and TRBC2 CAR T cells, show promising characteristics for utility potentially also as ADCs, offering another modality through which this targeting paradigm can be exploited for the treatment of peripheral T cell lymphomas. Citation Format: Mathieu Ferrari, Vania Baldan, Priyanka Ghongane, Alex Nicholson, Reyisa Bughda, Zulaikha Akbar, Patrycja Wawrzyniecka, Paul Maciocia, Shaun Cordoba, Simon Thomas, Shimobi Onuoha, Martin Pule. Targeting TRBC1 and 2 for the treatment of T cell lymphomas [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 2183.

Engineered T cell therapies show considerable promise in the treatment of refractory malignancies. Given the ability of engineered T cells to engraft and persist for prolonged periods along with unpredicted toxicities, incorporation of a suicide gene to allow selective depletion after administration is desirable. Rapamycin is a safe and widely available immunosuppressive pharmaceutical that acts by heterodimerization of FKBP12 with the FRB fragment of mTOR. The apical caspase caspase 9 is activated by homodimerization through its CARD domain. We developed a rapamycin-induced caspase 9 suicide gene. First, we showed that caspase 9 could be activated by a two-protein format with replacement of the CARD domain with both FRB and FKBP12. We next identified an optimal compact single-protein rapamycin caspase 9 (rapaCasp9) by fusing both FRB and FKBP12 with the catalytic domain of caspase 9. Functionality of rapaCasp9 when co-expressed with a CD19 CAR was demonstrated in vitro and in vivo.