Patent Application
20230381228
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 643662002740SEQLIST.TXT, date recorded: Oct. 29, 2021, size: 33,738 bytes).
The present disclosure relates generally to regulatory T cells (Tregs) engineered to express a dipeptidyl aminopeptidase-like protein 6 (DPP6)-reactive chimeric antigen receptor (CAR). The engineered Tregs are suitable for use in immunotherapy regimens for autoimmune, inflammatory and degenerative diseases.
Autoimmune diseases are a diverse collection of diseases arising as a consequence of attacks on one or more organs by an acquired immune response of an individual (e.g., autoantibody-mediated or self-reactive T cell-mediated). Autoimmune diseases are typically classified as systemic or organ-specific. Traditional treatments include immunosuppressants, such as non-steroidal anti-inflammatory drugs and glucocorticoids, which are administered to lessen the autoimmune response. Other treatments are administered to supplement or replace an organ-specific deficiency, but do not cure the underlying autoimmune diseases. For instance, diabetes mellitus can be well managed by subcutaneous injection of insulin. However, insulin injections do not achieve the tight glucose control of pancreatic islets, and therefore insulin therapy poses risks of complications from hyper- and hypoglycemia.
Thus, what is needed in the art are therapies for autoimmune diseases that are directed to inhibiting the underlying autoimmune response.
The present disclosure relates generally to regulatory T cells (Tregs) engineered to express a dipeptidyl aminopeptidase-like protein 6 (DPP6)-reactive chimeric antigen receptor (CAR). The engineered Tregs are suitable for use in immunotherapy regimens for autoimmune, inflammatory and degenerative diseases. In particular, the anti-DPP6 CAR expressing Tregs are suitable for treating or preventing autoimmune diseases of the pancreas or central nervous system.
The present disclosure relates generally to regulatory T cells (Tregs) engineered to express a dipeptidyl aminopeptidase-like protein 6 (DPP6)-reactive chimeric antigen receptor (CAR). The engineered Tregs are suitable for use in immunotherapy regimens for autoimmune, inflammatory and degenerative diseases. In particular, the anti-DPP6 CAR expressing Tregs are suitable for treating or preventing autoimmune diseases of the pancreas or central nervous system.
Tregs are a small subpopulation of peripheral blood lymphocytes and are critical for controlling tolerance, inflammation, and homeostasis of the immune system. Defects in Tregs have been observed in connection with uncontrolled inflammation and a variety of autoimmune diseases. Accordingly, Tregs are being developed as adoptive cell therapies for treating autoimmune and inflammatory diseases, graft-versus-host disease after bone marrow transplantation, and rejection of solid organ transplants (Bluestone and Tang, Science, 362:154-155, 2018).
In many autoimmune diseases, although Tregs are present, quantitative and/or qualitative defects result in an imbalance with disease-causing autoreactive effector T cells (Tconvs). Thus, restoring the balance between pathogenic Tconvs and Tregs could be a curative solution for many autoimmune diseases. Indeed, preclinical studies show therapeutic benefit of Treg infusion in a variety of autoimmune and inflammatory diseases. Importantly, for organ-specific autoimmune diseases and inflammation such as type I diabetes (T1D) and multiple sclerosis, Tregs with antigen specificity for the affected organ are often orders of magnitude more effective in halting disease. However, tissue-specific Tregs are often retained in the tissue and its draining lymph nodes, thus their frequency in the blood is very low making it difficult to isolate and expand Tregs for therapeutic use.
The past few years have witnessed exciting breakthroughs in cancer immunotherapy using T cells expressing a chimeric antigen receptor (CAR) targeting CD19. The feasibility of this approach to redirect polyclonal Tregs to a myriad of tissue antigens in pre-clinical models has also been reported. However, application of CAR technology to redirect Tregs to islet or brain antigens have not been described.
To select a CAR target for directing Tregs to pancreatic islets, proteins that are preferentially expressed in the islets were considered. Insulin is highly specific for pancreatic islets, but it is a soluble secreted protein. Although, multimeric soluble proteins can activate CARs and crystalized insulin stored inside granules may be able to trigger CARs, insulin crystals are rapidly solubilized and secreted crystalized insulin an unsuitable CAR target. Similarly, other islet hormones, such as glucagon, were also deemed to be unsuitable.
Cell surface proteins that are reported to be highly selective for pancreatic islets were considered to be viable CAR targets. Tetrapanin-7 (TSPAN7), calcium sensing receptor (CASR), prostaglandin D2 receptor 2 (PTGDR2), and dipeptidyl aminopeptidase-like protein 6 (DPP6) were selected for further assessment. Among these, CASR and PTGDR2 have multiple transmembrane and extracellular domains, and hence very complex structures, making it technically difficult to express these molecules as soluble proteins identification of binders in solution.
TSPAN-7 has three extracellular domains, one of which is large and readily expressed as a soluble protein. Initial screens in a Fab library identified several TSPAN7 binders. But unexpectedly, TSPAN-7 was found to be expressed on human B and T lymphocytes, eliminating this target from further consideration.
DPP6 is a single transmembrane protein with a larger extracellular domain. An initial screen yielded one Fab clone to DPP6. Around this time, the development of nanobodies targeting DPP6 for intravital imaging of beta cell mass was reported (Balhuizen et al., Scientific Reports, 7(1):15130, 2017). Four of the twelve anti-DPP6 (aDPP6) nanobodies, namely 2hD1, 2hD123-A24V, 2hD6 and 4hD29, were selected for CAR development based on their potential cross-reactivity with mouse DPP6 and levels of affinity.
Certain aspects of the present disclosure relate to CD4+, CD25+, CD127−/lo human regulatory T cells (Tregs) engineered to express a dipeptidyl aminopeptidase-like protein 6 (DPP6)-reactive chimeric antigen receptor (CAR) comprising an extracellular DPP6-binding domain linked through a hinge and a transmembrane domain to an intracellular domain comprising a costimulatory domain and an activation domain.
Dipeptidyl aminopeptidase-like protein 6, or “DPP6,” is a single-pass type II transmembrane protein that is also referred to as DPPX, VF2, MRD33, DPL1, dipeptidyl peptidase-like protein 6, or dipeptidyl peptidase IV-related protein. While DPP6 is a member of the peptidase S9B family of serine proteases, it does not display detectable protease activity. DPP6 is highly expressed in the human and mouse brain, and has been shown to bind specific voltage-gated potassium channels and alter their expression and biophysical properties. Variations in the DPP6 gene are associated with susceptibility to amyotrophic lateral sclerosis and with idiopathic ventricular fibrillation (Online Mendelian Inheritance in Man entry 126141; Ding et al., QJM, 111(6):373-37, 2018; and Brambilla et al., Neurosci Lett, 530(2):155-60, 2012). DPP6 has also been identified as a biomarker of endocrine cell mass that is detectable in the human pancreas (Balhuizen et al., Scientific Reports, 7(1):15130, 2017).
The DPP6 mRNA is subject to alternative splicing. In humans, there are eleven splice variants and eight protein isoforms of DPP6. The longest isoform of DPP6 is DPP6 isoform 1 (also referred to as DPP6 “L”). DPP6 isoform 1 is the DPP6 variant with the highest levels of expression in pancreatic islets (Balhuizen et al., Scientific Reports, 7(1):15130, 2017). The amino acid sequence of human DPP6 isoform 1 according to NCBI Reference Sequence NP_570629.2 is:
Isoform 1 is the dominant form of DPP6 expressed in pancreatic islets and the brain. The extracellular domain of isoform 1 includes residues 118-865 of SEQ ID NO:31. Isoforms 1, 2, 3 and 6 have identical extracellular domain, while isoform 4 has a small membrane-proximal truncation relative to Isoforms 1, 2, 3 and 6. In contrast, Isoform 5, 7, and 8 have very short extracellular domains. Additional information on DPP6 splice variants and protein isoforms, including nucleotide and amino acid sequence information, may be found in the NCBI Gene database, under Gene ID 1804.
The nanobodies described in Example 1 were made using a recombinant protein derived from the extracellular domain of isoform 1 as an immunogen. Thus, the nanobodies of Example 1 are expected to bind to isoforms 1, 2, 3 and 6, and possibly isoform 4, but not isoforms 5, 7 and 8. Likewise, the DPP6-binding domain of the DPP6-reactive CARs (aDPP6-CARs) of the Tregs of the present disclosure bind to the extracellular domain of isoform 1 (residues 118-865 of SEQ ID NO:31). In some embodiments, the DPP6-binding domain comprises a variable region of a DPP6-reactive nanobody. In other embodiments, the DPP6-binding domain comprises a DPP6-reactive scFv, or a DP66-reactive Fab. In some embodiments, the variable region of a DPP6-reactive nanobody comprises three complementarity-determining regions (CDRs) having amino acid sequences selected from: (i) a CDR1 of SEQ ID NO:13, a CDR2 of SEQ ID NO:14, and a CDR3 of SEQ ID NO:15; (ii) a CDR1 of SEQ ID NO:16, a CDR2 of SEQ ID NO:17, and a CDR3 of SEQ ID NO:18; (iii) a CDR1 of SEQ ID NO:19, a CDR2 of SEQ ID NO:20, and a CDR3 of SEQ ID NO:21; and (iv) a CDR1 of SEQ ID NO:22, a CDR2 of SEQ ID NO:23, and a CDR3 of SEQ ID NO:24. In some embodiments, the variable region comprises the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, or an amino acid sequence sharing at least 90%, 95% or 99% identity with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the variable region of the DPP6-reactive nanobody comprises one or more conservative amino acid substitution(s). In some embodiments, the conservative amino acid substitution(s) are located in a framework region of the DPP6-reactive nanobody. In other embodiments, the conservative amino acid substitution(s) are located in a CDR of the DPP6-reactive nanobody. In further embodiments, the conservative amino acid substitution(s) are located in both a framework region and a CDR of the DPP6-reactive nanobody.
The hinge of the aDPP6-CARs of the Tregs of the present disclosure connects the DPP6-binding domain to the transmembrane domain. In some embodiments, the hinge comprises an IgG4 hinge. In some embodiments, the hinge further comprises the CH3 domains of IgG4, or both the CH2 and CH3 domains of IgG4. In circumstances in which the hinge comprises the CH2 domain of IgG4, the CH2 domain may comprise one or both of L235E and N297Q substitutions. In other embodiments, the hinge comprises a CD28 hinge, or a CD8a hinge.
In exemplary embodiments, the transmembrane domain of the aDPP6-CARs of the Tregs of the present disclosure is a CD28 transmembrane domain. In other embodiments, the transmembrane domain is a CD8a transmembrane domain.
The intracellular domain of the aDPP6-CARs of the Tregs of the present disclosure comprises a costimulatory domain and an activation domain. In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain. In some embodiments, the activation domain comprises a CD3 activation domain. In some embodiments, the CD3 activation domain comprises a CD3 zeta activation domain. In other embodiments, the CD3 activation domain comprises a CD3 epsilon activation domain, a CD3 delta activation domain or a CD3 gamma activation domain.
The anti-DPP6 CAR Tregs of the present disclosure are suitable for use in methods of treating or preventing a pathological immune response in a human subject in need thereof. In some embodiments, the pathological immune response presents as an autoimmune disease, such as an autoimmune disease of the pancreas or central nervous system. In other embodiments, the pathological immune response presents as a neurodegenerative disease. References and claims to methods comprising administering an effective amount of anti-DPP6 CAR Tregs or a pharmaceutical composition thereof to a human subject, in their general and specific forms likewise relate to:
In some embodiments, the effective amount of anti-DPP6 CAR Tregs or the pharmaceutical composition comprises from 105 to 1011 of the human Tregs. That is, an effective amount comprises greater than or equal to 105, 106, 107, 108, 109, or 1010 Tregs, and less than or equal to 1011, 1010, 109, 108, 107, or 106 Tregs. In some embodiments, the effective amount of anti-DPP6 CAR Tregs or the pharmaceutical composition is administered to the human subject by intravenous infusion over an interval of from 1 to 120 minutes. That is, an effective amount is infused intravenously in an interval greater than or equal to 1, 2, 3, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90 or 105 minutes, and less than or equal to 120, 05, 90, 75, 60, 45, 30, 25, 20, 15, 10, 5, 4, 3 or 2 minutes. In other embodiments, the effective amount of anti-DPP6 CAR Tregs or the pharmaceutical composition is administered to the human subject locally in conjunction with pancreatic islet or beta cell replacement therapy.
Certain aspects of the present disclosure relate to methods for the production of recombinant human regulatory T cells (Tregs) engineered to express a dipeptidyl aminopeptidase-like protein 6 (DPP6)-reactive chimeric antigen receptor (CAR), comprising:
In some embodiments, the nucleic acid encoding the DPP6-reactive CAR is introduced into the human Tregs by transfection. In other embodiments, the nucleic acid encoding the DPP6-reactive CAR is introduced into the human Tregs using lentiviral transduction. In some embodiments, the nucleic acid encoding the DPP6-reactive CAR is introduced into the human Tregs using a CRISPR engineering system.
As used herein and in the appended claims, the singular form “a,” “an” and “the” includes plural forms unless indicated otherwise. For instance, “an” excipient includes one or more excipients.
The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of” is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of” is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments. It is understood that aspects and embodiments described herein as “comprising” include “consisting of” and “consisting essentially of” embodiments.
The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value (e.g., about 200 fold refers to 180 fold to 220 fold and includes 200 fold).
As used herein, numerical ranges are inclusive of the numbers defining the range (e.g., 10 to 20 amino acids encompasses 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 amino acids.
Exemplary amino acid sequences are set forth in sequence identifiers throughout the present disclosure. Some of the claimed embodiments are described by reference to a percent identity shared with an exemplary amino acid sequence. Two amino acid sequences are substantially identical if their amino acid sequences share at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a specified region, or, when not specified, over their entire sequences), when compared and aligned for maximum correspondence over a comparison window or designated region. As pertains to the present disclosure and claims, the BLASTP sequence comparison algorithm using default parameters is used to align amino acid sequences for determination of sequence identity.
Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., J Mol Biol, 215: 403-410, 1990; and Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).
As used herein, the term “isolated” refers to an object (e.g., Tregs) that is removed from its environment (e.g., separated). “Isolated” objects are at least 50% free, preferably 75% free, more preferably at least 90% free, and most preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) free from other components with which they are associated.
An “effective amount” of an agent disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” may be determined empirically in relation to the stated purpose. An “effective amount” or an “amount sufficient” of an agent is that amount adequate to affect a desired biological effect, such as a beneficial result, including a beneficial clinical result. The term “therapeutically effective amount” refers to an amount of an agent (e.g., human Tregs) effective to “treat” a disease or disorder in a subject (e.g., a mammal such as a human). An “effective amount” or an “amount sufficient” of an agent may be administered in one or more doses.
The terms “treating” or “treatment” of a disease refer to executing a protocol, which may include administering one or more drugs to an individual (human or otherwise), in an effort to alleviate a sign or symptom of the disease. Thus, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a palliative effect on the individual. As used herein, and as well-understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission. “Treatment” can also mean prolonging survival of a recipient of an allograft as compared to expected survival of a recipient of an allograft not receiving treatment. “Palliating” a disease or disorder means that the extent and/or undesirable clinical manifestations of the disease or disorder are lessened and/or time course of progression of the disease or disorder is slowed, as compared to the expected untreated outcome.
As used herein, the term “pathological immune response” encompasses autoimmune diseases, and autoinflammatory diseases. “Autoimmune diseases” involve immune recognition resulting in direct damage to self-tissue and functional impairments. Pathologically, autoimmune diseases are typically driven by cells of the adaptive immune system. An example of an autoimmune disease is type I diabetes. “Autoinflammatory diseases” involve spontaneous activation, or over-reaction of the immune system to non-self-antigens (e.g., environmental, food, commensal or other antigens) resulting in indirect (bystander) damage to self-tissue and functional impairments. Pathologically, autoinflammatory diseases are typically dominated by cells of the innate immune system. An example of an autoinflammatory disease is the neurodegenerative disease known as Lou Gehrig's disease or amyotrophic lateral sclerosis.
The present disclosure is described in further detail in the following examples, which are not intended to limit the scope of the disclosure as claimed. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure.
In the experimental disclosure which follows, the following abbreviations apply: Ab (antibody); CAR (chimeric antigen receptor); DPP6 (dipeptidyl aminopeptidase-like protein 6); FACS (fluorescence-activated cell sorting); HLA (human leukocyte antigen); IEQ (islet equivalent); IL-2 (interleukin-2); MFI (mean fluorescent intensity); MOI (multiplicity of infection); NSG (NOD SCID Gamma); PBMC (peripheral blood mononuclear cell); SBC (stem cell-derived beta cells); STII (Streptavidin Tag II); STZ (streptozotocin); Tconv (conventional T cell); Teff (effector T cell); Treg (regulatory T cell); TSDR (Treg-specific demethylation region); and UCSF (University of California San Francisco).
This example describes the generation of anti-DPP6 CAR constructs, as well as human T cells engineered to express the anti-DPP6 CARs (aDPP6 CAR T cells).
Materials and Methods
Generation of anti-DPP6 CAR constructs. The anti-DPP6 nanobodies 2hD1, 2hD123-A24V, 2hD6 and 4hD29 were used to generate anti-DPP6 CAR constructs. DNA sequences corresponding to each nanobody followed by a tag (e.g., C-terminal myc tag) were synthesized. Each DNA sequence was further ligated following the Gibson assembly protocol to a pre-digested in-lab vector containing an IgG4 hinge, CD28 transmembrane and intracellular domains, a CD3zeta intracellular domain, a P2A self-cleaving peptide and mCherry DNA sequences (Table 1-1), as well as the ampicillin resistance gene. Competent E. coli were transformed with the different ligation products. Five colonies/plate were picked and overnight cultures were grown for DNA purification. Successful transformation and ligation was confirmed by performing DNA sequencing of the ligation products. The CAR constructs were further cloned into a lentiviral vector with generation 2 backbone. The amino acid sequences of the CAR domains and anti-DPP6 nanobodies are provided in Table 1-1 and Table 1-2. In Table 1-3, the amino acid sequence shared by the four CARs is set forth as SEQ ID NO:25 (IgG4 Hinge+CD28 TM+CD28 endo+CD3z), while the amino acid sequences of the mature CARs (absent signal peptide, P2A and mCherry) are set forth as SEQ ID NOs:27-30.
Transduction of anti-DPP6 CARs constructs into primary human T cells. Heparinized venous blood was collected from healthy donors, diluted 1:1 with PBS and then layered on a density gradient (Ficoll). Peripheral blood mononuclear cells (PBMCs) were collected from the interface after centrifugation, washed with PBS+FBS2%, and resuspended in PBS+FBS2%+EDTA2 mM. CD4+ Tconvs and Tregs were purified after negative enrichment of CD4+ cells (EasySep™ Human CD4+ T Cell Isolation Kit, StemCell), followed by CD4, CD25 and CD127 staining and cell sorting of CD4+ CD25low/−CD127hi cells (Tconvs) and CD4+ CD25hi CD127low cells (Tregs) using the BD FACSAria II. Human CD4+ Tconvs and Tregs were stimulated for 48 hours with aCD3/aCD28 Dynabeads at 1:1 ratio and cultured in complete RPMI supplemented with IL2 (100 IU/ml for Tconvs, 300 IU/ml for Tregs). Cells were next spin-fected by adding virus at an MOI of 1:1 and polybrene at 5 ug/ml final. The culture was then checked every other day and fresh complete RPMI supplemented with IL2 was added when needed. CAR transduction efficiency and membrane expression were evaluated 5 days after transduction by assessing intracellular expression of mCherry protein and membrane expression of the Tag (
Anti-DPP6 CAR activation of CD4+ Tconvs by human islets in vitro. Pancreas from deceased non-diabetic donors was harvested and digested. Islets were hand-picked and put in MIAMI medium. Islets were then enzymatically dissociated (Accumax) and washed in MIAMI medium. After counting, islet cells were resuspended at 1×106 cells/ml in MIAMI medium and 100 ul/well of the cell suspension were dispatched to wells of a 96-well round bottom plate. mCherry/CAR+ Tconvs (CD4+CD25low/−CD127hi cells) were sorted 7 days after transduction and kept in culture with complete RPMI+IL2 (100 IU/ml) for 3 more days. Cells were then used fresh, or frozen and thawed when islets were available. Tconvs were cultured for 24 hours with complete RPMI+IL2 prior to co-culture with islets. CAR+ Tconvs were resuspended at 1×106 cells/ml in MIAMI medium and 100 ul/well of the cell suspension were added to wells of the 96-well round bottom plate containing the dissociated islets. Cells were co-cultured for 48 hours at 37° C. At the end of the culture, Tconv activation was assessed by staining for CD71, ICOS and CD25. Samples were acquired on a BD FortessaX20 cytometer and analyzed using the FlowJo software. Untransduced and anti-HLA-A2 CAR-transduced CD4+ Tconvs from the same donor were used as controls (
Anti-DPP6 CAR activation of CD4+ Tregs by human islets in vitro. Pancreas from deceased non-diabetic donors was harvested and digested. Islets were hand-picked and put in MIAMI medium. Islets were then enzymatically dissociated (Accumax) and washed in MIAMI medium. After counting, islet cells were resuspended at 1×106 cells/ml in MIAMI medium and 100 ul/well of the cell suspension were dispatched to wells of a 96-well round bottom plate. mCherry/CAR+ Tregs (CD4+CD25hi CD127low/− cells) were sorted 7 days after transduction and kept in culture with complete RPMI+IL2 (300 IU/ml)+aCD3/aCD28 Dynabeads (1:1 ratio) for 3 more days. Cells were then used fresh, or frozen and thawed when islets were available. Tregs were cultured for 24 hours with complete RPMI+IL2, without TCR stimulation, prior to co-culture with islets. CAR+ Tregs were resuspended at 1×106 cells/ml in MIAMI medium plus IL2 and 100 ul/well of the cell suspension were added to wells of the 96-well round bottom plate containing the dissociated islets. Cells were co-cultured for 48 hours at 37° C. At the end of the culture, Treg activation was assessed by staining for CD71, ICOS and CD25. Samples were acquired on a BD FortessaX20 cytometer and analyzed using the FlowJo software. Untransduced and anti-HLA-A2 CAR-transduced Tregs from the same donor were used as controls (
Administration of anti-DPP6 CAR expressing CD4+ Tconvs to mice. Streptozotocin (STZ) was injected into NSG mice (216 mg/kg) to deplete endogenous mouse islets. Once mice became diabetic (i.e. glycemia>300 mg/dl and ketone detected in the blood) 3,000IEQ human islets were transplanted into the kidney capsule, and glucose levels were monitored every other day. Once mouse glycemia was stably normalized, 0.8×106 CAR+ or polyclonal CD4+ Tconvs were intravenously injected and glucose levels were monitored every other day (
Administration of anti-DPP6 CAR expressing Tregs to mice. STZ was injected into NSG mice and 3,000IEQ human islets were transplanted into the right kidney capsule once mice were diabetic, as described above. Glucose levels were monitored every other day. Once mouse glycemia was stably normalized, 1 to 2×106 CAR+ or polyclonal luciferase-expressing Tregs were intravenously injected at day 40 and 25,000U/mouse of IL2 was administered twice a day for 8 days. Glucose monitoring was performed every other day. At day 73, nephrectomy of the kidney transplanted with human islets was performed on all mice, and glucose levels were monitored every day (
Assessment of anti-DPP6 CAR-expressing CD4+ Tconvs in vivo binding to mouse DPP6. Human CD4+ Tconvs were transduced to express luciferase and anti-HLA-A2 CAR or an anti-DPP6 CARs (2hD1 or 2hD6 clones), as previously described. Five days after transduction, aCD3/aCD28 Dynabeads were removed, transduction assessed and 2 days later anti-DPP6 CAR-expressing Tconvs were sorted. One week later, the different CD4+ Tconv cultures were counted and injected intravenously at the following doses: 2 or 4 million cells of polyclonal CD4+ Tconvs; 4 million aHLA-A2 CAR CD4+ Tconvs; 1.5 million (2hD-1) or 2.5 million (2hD-6) aDPP6 CAR CD4+ Tconvs. Bioluminescence imaging was performed 2, 4, 6 and 9 days after T cell injection. After the last bioluminescence imaging, mice were sacrificed and the spleen, lung, brain, liver, spinal cord and leg bone were harvested, put in a bath of diluted luciferin, and imaged. Organ specimens were also saved to perform further immunofluorescent stainings.
To generate anti-DPP6 CARs, the anti-DPP6 nanobodies 2hD1, 2hD123-A24V, 2hD6 and 4hD29 were selected based on their potential cross-reactivity with mouse DPP6 and their various levels of affinity (Table 1-4). Binding of the nanobodies to primary human islets was verified using flow cytometry (
The nanobodies were used to generate anti-DPP6 CAR constructs (
To evaluate the in vitro functionality of the anti-DPP6 CAR-expressing human T cells, the engineered T cells were co-incubated with primary dissociated human islets, and T cell activation was assessed. Specifically, anti-DPP6 CAR-expressing CD4+ Tconvs (
To further evaluate the in vitro functionality of the anti-DPP6 CAR-expressing human T cells, the engineered T cells were cultured in the presence and absence of human SBC for 48 hours before T cell activation was assessed by flow cytometry. The expression of activation markers (CD71, ICOS, CD25) by polyclonal and anti-DPP6 CAR-expressing CD4+ conventional T cells is shown in Table 1-5. The expression of activation markers by polyclonal and anti-DPP6 CAR-expressing CD4+ regulatory T cells is shown in Table 1-6. Thus, anti-DPP6 CAR expressing CD4+ Tconv and Tregs get specifically and robustly activated by SCB in vitro.
Next, the anti-DPP6 CAR-expressing human T cells were administered to mice in order to test whether the T cells would migrate to and be activated by human islets in vivo. Specifically, anti-DPP6 CAR-expressing CD4+ Tconvs (
Of the mice injected with Tconvs, a rapid and strong increase in glycemia was observed less than 10 days after T cell injection only in the group of mice that received anti-DPP6 CAR-expressing Tconvs (
In the Treg administration experiment, for more than 1 month after the CAR Treg injection the mice remained normo-glycemic. To ensure that this observation was not due to a rebound of mouse islets, a nephrectomy of the kidney transplanted with human islets was performed on all mice and glucose levels were monitored daily. Shortly after nephrectomy, a rapid and substantial increase in glycemia was observed in all the animals. This observation confirmed not only the lasting functionality of the transplanted human islets, but also the absence of toxicity of the injected CAR Tregs.
In parallel, the in vivo migration of the CAR Tregs was evaluated by bioluminescence. While the anti-HLA-A2 CAR-expressing Tregs accumulated within a few days in the kidney transplanted with the human islets, anti-DPP6 CAR-expressing Tregs took longer to do so. Indeed, the bioluminescence signal remained wide spread for more than one week, with most luminescence seen around the spinal cord and the brain tissue where mouse DPP6 is expressed. This demonstrates that while anti-DPP6 nanobody had not been reported to cross the brain blood barrier, anti-DPP6 CAR Tregs can cross into the central nervous system. The ability of anti-DPP6 CAR-expressing Tconvs to interact with mouse DPP6 in vivo was also assessed. Specifically, CAR-expressing Tconvs that also express luciferase were injected into mice and visualized using bioluminescent imaging in vivo or ex vivo in isolated tissues. While polyclonal and anti-HLA-A2 CAR Tconvs gave a brief signal in the spleen before vanishing, a bright and persistent signal around the tissues of the central nervous system was observed in the mice injected with anti-DPP6 CAR expressing Tconvs. When individual tissues were imaged, signal in the brain was detected only in the mice injected with anti-DPP6 CAR-expressing Tconvs, confirming the potential of anti-DPP6 CAR to cross-react with mouse DPP6.
To determine the effect of the location of the Myc tag on detection of CAR expression on T cells, expression of a construct in which the Myc tag was placed at the N-terminal end of the 2hD123-A24V nanobody (nMyc) was compared to expression of a construct in which the Myc tag was placed at the C-terminal end of the 2hD123-A24V nanobody (cMyc). Schematic diagrams of the anti-DPP6-cMyc CAR and the anti-DPP6-nMyc CAR are shown in
In conclusion, anti-DPP6 CAR-expressing Tregs and Tconvs were generated, and determined to be capable of being specifically activated by human islet cells both in cell culture, and in vivo in a mouse model. Additionally, anti-DPP6 CAR-expressing Tregs and Tconvs were determined to be capable of being specifically activated by human stem cell-derived beta cells (SCB) in vitro.
This application claims benefit of U.S. Provisional Application No. 63/107,110, filed Oct. 29, 2020, the disclosure of which is hereby incorporated by reference in its entirety.
This invention was made with government support under Grant No. UC4 DK116264 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/72139 | 10/29/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63107110 | Oct 2020 | US |
