Compositions and methods for treating cancer with anti-cd33 immunotherapy
A technology for antigens and uses, applied in the field of chimeric antigen receptors
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Embodiment 1
[0317] Example 1. Isolation of CD33-specific antibodies from fully human phage-displayed ScFv and VH sub-libraries
[0318] Materials and methods:
[0319] a) Production of human phage-displayed ScFv and VH CD33-specific antibodies
[0320] A naive human ScFv (recombinant single-chain variable fragment of immunoglobulin) phage display library (approximately diverse, with 10 10 unique specificity) (Z.Y. Zhu and D.S. Dimitrov, unpublished data) and human HV (immunoglobulin heavy chain variable domain) libraries to select recombinant human CD33-specific ScFv or VH. will be 10 12 Amplified libraries of phage-displayed ScFv or VH were mixed with 5, 3 and 1 μg evenly distributed in 5 x 100-μl volumes in 5 wells of a 96-well plate during the first, second and third rounds of biopanning, respectively. Coated CD33 was incubated for two hours at room temperature. After each round of incubation, the wells were washed 5 times for the first round with phosphate-buffered saline (PBST) c...
Embodiment 2
[0327] Example 2. Expression of an anti-CD33 CAR based on a fully human heavy chain-only Ig or scFv-based binding sequence
[0328] In this example, novel fully human immunoglobulin heavy chain-only anti-CD33 CAR T cells derived from single-chain variable fragment (scFv) conjugate sequences are described. Novel anti-CD33 CART constructs have demonstrated high-level expression in primary human T cells and have specific and potent cytotoxic and cytokine functions against CD33-positive tumor cells.
[0329] Homo sapiens CD33 (sialic acid binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67) is a well-studied target on acute myeloid leukemia (AML). CD33 humanized antibodies (lintuzumab) and CD33 antibody-drug conjugates (gemtuzumab or GO, Pfizer) have shown some efficacy but failed to show robust efficacy in clinical trials Therapeutic benefit (1. Feldman EJ et al., J Clin Oncol 2005;23(18):4110-4116, 2. Petersdorf SH et al., Blood 2013;121(24):4854-4860). AMG330, a CD33-CD3 b...
Embodiment 3
[0366] Example 3. Improved functional properties of the CD33 CAR portion can be achieved by altering the structure of an anti-CD33 CAR expressing a fully human heavy chain-only or ScFv-based binding sequence
[0367] In this example, different structural configurations of anti-CD33 CAR T cells derived from novel fully human immunoglobulin heavy chain-only or scFv binder sequences are described.
[0368] When challenged with tumor cells expressing antigen, for example by incorporating a single CD28-derived vs CD137 / 4-1BB-derived co-stimulatory domain in frame with an activation domain (e.g., CD3ζ) (2nd generation CAR), lack of co-stimulatory domain CAR structures with stimulatory domains (1st generation CARs) or incorporating multiple co-stimulatory domains in tandem (3rd generation CARs), assuming that CAR T cells secrete higher or lower levels of inflammatory cytokines such as IL-2 , IFNγ, TNFα.
[0369] In some constructs, the incorporation of novel hinge and transmembrane ...
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