Introduction
Chimeric antigen receptor T cell (CART) products targeting CD19 have been approved for the treatment of patients with relapsed and/or refractory (r/r) B cell malignancies [
1‐
4], including patients with acute lymphoblastic leukemia (ALL) [
2,
3].
All commercially available CARTs express second-generation CARs that contain one costimulatory domain. Third-generation CARTs comprising two costimulatory domains have shown superior engraftment, improved expansion capacity and prolonged persistence [
5‐
9]. Clinically, superior expansion and longer persistence were observed when CD19-directed second (CD28 costimulatory domain)- and third-generation (CD28 and 4-1BB) CARTs were simultaneously administered to lymphoma patients [
10]. However, clinical data evaluating solely third-generation CARTs are limited [
11].
Here, we describe the first results obtained with an academically developed third-generation CAR in the framework of the investigator-initiated trial (IIT) Heidelberg CAR number 1 (HD-CAR-1) in adult patients with r/r ALL. All steps of treatment including leukapheresis, manufacturing and administration of CARTs, patient monitoring as well as patient follow-up were performed in-house.
A solely academic-driven trial, a third-generation Good Manufacturing Practice (GMP) grade retroviral vector and treatment of adult r/r ALL patients with escalating CART doses make this trial hitherto unique.
Discussion
Despite novel therapeutic options for treatment of r/r ALL patients [
27‐
29], the outcome for older patients remains poor [
30‐
32]. Here, we treated adult ALL patients with escalating doses of CD19-specific third-generation CARTs. For all patients, CART products were successfully manufactured. Despite low CART doses administered and a heavily pre-treated patient cohort, a CR rate of 80% including 50% MRD-negative CR was achieved. At 12 months, 38% of evaluable patients remained progression-free and median OS was not reached. Patients who achieved an initial MRD-negative CR did not reach median PFS and were all alive at 1-year follow-up.
The CAR construct used within HD-CAR-1 has already been evaluated in the context of two clinical trials that focused on patients with non-Hodgkin’s lymphoma (NHL). Also, five adult ALL patients (
n = 1 [
10],
n = 4 [
11]) were included. No response in the single ALL patient [
10] and CR in 50% (two of four ALL patients) [
11] were reported, although results were limited by the small patient number.
Result of HD-CAR-1 is in line with previous trials of second-generation CARTs in adult ALL patients: Park et al. reported a CR rate of 83% (
n = 53), with 50% OS and 30% event-free survival (EFS) one year after treatment [
33]. In the ZUMA-3 trial resulting in the approval of Brexucabtagene autoleucel for treatment of adult r/r ALL patients in 2021, a CR rate of 71% (
n = 55) with 12-month OS of 75% and EFS of 50% was observed. These results might be at least in part be attributable to the strict exclusion criteria in the ZUMA-3 trial of included patients [
34]. Frey et al. reported a CR rate of 69% (
n = 35) with OS of 47% and EFS of 31% at two-year follow-up. In this latter trial, high-dose (5 × 10
8), fractionated CART administration (
n = 20) resulted in unmet median OS and EFS one year after treatment. Of note, only 25% of patients had received a prior alloSCT [
35].
Relapses post-CARTs have been reported in 30 to 60% of ALL patients [
33,
36‐
39]. Also in HD-CAR-1, 50% of patients with CR at EOS, have relapsed in the first year after treatment and three patients have received a consecutive alloSCT after HD-CAR-1 treatment. In line with data on the ZUMA-3 trial [
34], efficacy of HD-CAR-1 CARTs was limited in patients with extramedullary disease: Only one patient (UPN #13) with extramedullary disease at CART treatment did not relapse after CART treatment, underlining the difficulties of treatment of this patient subgroup.
Of note, HD-CAR-1 appears to be associated with a highly favorable toxicity profile, even at high-dose levels: No ICANS or higher-grade CRS occurred, and only low-grade (I-II°) CRS was observed in 31% of the patients. Prior alloSCT was associated with fatal exacerbation of a most likely preexisting GvHD in one patient. Although an immunogenic effect of CARTs cannot be excluded, GvHD might have been rather triggered substantially by preceding therapies including inotuzumab ozogamicin and blinatumomab.
As for hematotoxicity, the rate of prolonged neutropenia was comparable or lower than the one of previous reports [
37,
40,
41], despite the high rate of patients after an alloSCT.
CD28-costimulation has been associated with rapid expansion and marked anti-tumor efficacy [
42‐
44], and 4-1BB has been shown to enhance proliferation, to reduce exhaustion and to mediate long-term CART persistence [
45‐
47]. In fact, we observed fast expansion of HD-CAR-1-CARTs. In contrast to loss of CARTs in the PB of patients treated in ZUMA-3 on day 28 [
48], CARTs were durably detected in HD-CAR-1 patients. In line with others, initial MRD-negative response [
38,
49] and higher doses of administered CARTs [
35] resulted in higher CART frequencies and improved outcome.
High-resolution immunophenotyping revealed an immune cell repertoire of responders characterized by general activation of T cells. In contrast to others, we observed no influence on response by myeloid subtypes [
11] or CD4 + /CD8 + T cell ratio [
50,
51]. Interestingly, the patient with the most durable response to treatment (UPN#11) displayed a distribution of immune cells in his PB which resembled the cellular composition of the PB of healthy donors. In patients responding to CART therapy, we observed a higher number of γδ T cells within collected PBMCs. In fact, infiltration of malignancies with γδ T cells is associated with favorable prognosis [
52] and in the allo-SCT setting, γδ T cells have been associated with enhanced anti-tumor response, improved OS and reduced occurrence of GvHD [
53,
54].
Within the CART product, expression of CD39 on effector T cells predicted response: Low levels of this T cell subset were observed in responders, high levels in non-responders. CD39 is expressed on T cell subsets [
55,
56], and its expression on CD8-positive T cells has been associated with T cell exhaustion [
57,
58]. While CARTs with a less differentiated phenotype, e.g., central memory or naïve CARTs, mediate better expansion, persistence and antitumor activity [
59,
60], T cell exhaustion is associated with inferior response [
61,
62]. In the context of CARTs, CD39 expression has been linked to reduced CART expansion [
63,
64]. Here, we confirm clinically that CD39 within the CART product might be highly relevant to predict outcome in CART patients. In contrast to other molecules such as PD-1 that have been identified not only in CART samples but also in healthy individuals [
65], CD39 might constitute a more specific marker for T cell exhaustion.
Declarations
Competing interests
AS: Travel grants from Hexal and Jazz Pharmaceuticals. Research grant from Therakos/Mallinckrodt. Consultancy BMS, Janssen-Cilag. Co-founder and part-time employee of TolerogenixX LtD. of TolerogenixX Ltd. CMT: research support from Bayer AG. Advisory board member Pfizer, Janssen-Cilag GmbH. Grants and/or provision of investigational medicinal products from Pfizer, Daiichi Sankyo, BiolineRx. FL: Advisory roles for Novartis, Incyte, Sanofi Aventis and Bristol-Myers Squibb. GB: Research support from Novartis; Consultancy for Novartis, Pfizer, Gilead, Celgene; Honoraria from Jazz, Celgene, Gilead; Travel support from Neovii, Jazz, Gilead. JC: Travel grants: Pfizer, Ipsen, Medac; Adboard: Pfizer, Merck, Ipsen, MSD; Consultancy: Pfizer, Merck, Medac. JK: Consultancy Novartis, Global Blood Therapeutics, bluebird bio. MB: consulting fees from Amgen and PRMA, research funding from Amgen, honoraria/travel grants from Jazz, Celgene, Novartis, Pfizer and Amgen, advisory board member for Incyte and Amgen. MLS: consultancy for Kite/Gilead, Takeda. MS: research grants from Apogenix, Hexal and Novartis. Travel grants from Hexal and Kite. Financial support for educational activities and conferences from bluebird bio, Kite and Novartis. Advisory board member of MSD. (Co-)PI of clinical trials of MSD, GSK, Kite and BMS. Co-Founder and shareholder of TolerogenixX Ltd. PD: consultancy AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche; speakers bureau AbbVie, Gilead, Novartis, Riemser, Roche; research support from Neovii and Riemser. None of the mentioned sources supported the work described within this manuscript. PDe: honorarium from MSD. PWu: Research support from the German Red Cross Blood Service Baden-Württemberg – Hessen gGmbH. Advisory Board Member of Sanofi-Aventis. TS: Consultant AbbVie, Takeda, Astellas, Amgen, Bristol-Myers Squibb, Gilead, Ridgeline Discoveries. Honorarium: Pfizer, AbbVie, Jazz Pharmaceuticals. Financial support congress participation: AbbVie, Jazz Pharmaceuticals., ADH, AHK, AKei, AKul, AKun, AN, BM, BN, DH, DV, DW, FK, JF, LJS, LW, MG, MRi, NG, PP, PW, SH, SLa, SY: none.
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