A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) remains a challenging pediatric and adult disease. Given the elevated expression of the CD33 antigen on leukemic blasts, therapeutic approaches to AML now feature the approved antibody drug conjugate (Mylotarg, GO) and investigational CART cell approaches incorporating CD33-binding domains derived from humanized scFvs. We designed a functional chimeric antigen receptor utilizing a human targeting sequence, derived from a heavy chain variable domain, termed CAR33VH. Lentiviral-based expression vectors which encoded CAR constructs incorporating the novel binding domain (CAR33VH), or the My96 scFv control binder (My96CAR) in frame with a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3 zeta activation domain, were transduced into primary human CD4+ and CD8+ T cells, and CAR expression was confirmed by flow cytometry. CAR33VH, similar to My96CAR, demonstrated robust and specific cytotoxicity in short-term and long-term co-incubation killing assays against CD33+ AML lines. In overnight cytokine release assays in which CAR T cells were challenged with the CD33+ tumor cells HL-60, MOLM-14 and KG-1a, CAR33VH elicited IFN-gamma, TNF-alpha and IL-2. This was seen with CD33+ cell lines, but not when CAR T were cultured alone. Studies with a CD33− cell line engineered to stably express the full length CD33 variant 1, or the naturally occurring CD33 splice variant 2, revealed that both CAR33VH and My96CAR, target the V domain of CD33, suggesting a similar therapeutic profile. Colony-formation assays utilizing peripheral blood CD34+ hematopoietic stem cells treated with CAR33VH, My96CAR, or with an untransduced T cell control, yielded similar numbers of BFU-E erythroid and CFU-GM myeloid colonies, suggesting a lack of CAR-related overt toxicity. In an in vivo AML model, NSG mice engrafted with MOLM-14 cells stably expressing firefly luciferase, both CAR33VH and CARMy96 efficiently eliminated tumors. In conclusion, we demonstrate for the first time the feasibility and efficacy of employing human variable domain-only binder derived from a phage display library in an anti-AML CAR design. CAR33VH, comprised of a human heavy-chain variable fragment-only antigen binding domain, was efficient in tumor killing in vitro and in vivo, and showed comparable functionality to the scFv-based My96CAR.

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains

“…Immunoglobulin-like molecules with antigen-binding domains made up of only heavy chains without light chains were first described in camelids and cartilaginous fish [25][26][27][28] . Binding domains made up of only a single immunoglobulin heavy-chain variable region domain have been reported to exhibit strong and specific antigen binding 25,[29][30][31][32][33] . Unlike a scFv, single heavychain-only binding domains have no need for a potentially immunogenic linker to connect the heavy chain to the light chain.…”

Fully human single-domain antibodies against SARS-CoV-2

“…1a). Furthermore, we and others have previously identified some isolated human VH single domains, which were independently folded and exhibited very similar biophysical properties to camelid nanobodies 16,17 . These findings inspired us to revisit the structural feature of single-domain antibodies, and hypothesize that certain VH framework regions could compensate for the absence of VL, resulting in the soluble human single-domain antibodies.…”

Antigen-specific stimulation and expansion of CAR-T cells using membrane vesicles as target cell surrogates

“…Synthetic CAR19 gene was purchased (GeneCust) and cloned into the pLV2 lentiviral vector (Clontech) under control of the EF1a promoter. The nucleotide sequences of the previously published CD33 scFv (clone my96 23 ) and CD123 (clone Klon43 or 32716 24 ) scFv were synthesized and inserted into the pLV2 CAR backbone to replace anti-CD19 scFv. To obtain IL-13-CAR construct, the DNA fragment coding for the IL-2 leader peptide, IL13 mutein (E13Y), human IgG4-CH2CH3 domain, CD28 transmembrane domain, and costimulatory domains of CD28, OX-40, and the CD3ζ-chain was synthesized (GeneCust) as a single cDNA and cloned into the pLV2 lentiviral vector (Clontech) as described for CD19 CAR.…”