Conditionally active chimeric antigen receptors for modified t-cells

Abstract

This disclosure relates to a chimeric antigen receptor for binding with a target antigen. The chimeric antigen receptor comprises at least one antigen specific targeting region including a multispecific antibody evolved from a wild-type antibody or a fragment thereof and having at least one of: (a) a decrease in activity in the assay at the normal physiological condition compared to the wild-type antibody or the fragment thereof, and (b) an increase in activity in the assay under the aberrant condition compared to the wild-type antibody or the fragment thereof. A method for using the chimeric antigen receptor and cytotoxic cells for cancer treatment is also provided. A method for producing the chimeric antigen receptor is also provided.

Description

RELATED APPLICATION DATA

[0001] This application claims benefit of U.S. provisional application No. 62 / 043,067, filed on Aug. 28, 2014, now expired, and international application no. PCT / US15 / 47197, filed on Aug. 27, 2015, and which designates the United States of America, both of which applications are hereby incorporated by reference in their entirety.FIELD OF THE DISCLOSURE

[0002] This disclosure relates to the field of protein evolution. Specifically, this disclosure relates to a method of generating a conditionally active chimeric antigen receptor from a wild type protein. The conditionally active chimeric antigen receptor is reversibly or irreversibly inactivated at a wild type normal physiological condition, but is active at an aberrant condition.BACKGROUND OF THE DISCLOSURE

[0003] There is a considerable body of literature describing the potential for evolving proteins for a variety of characteristics, especially enzymes. For example, enzymes may be evolved to be stabilized for op...

Examples

example 1

Generation of scFv Conditionally Active Antibodies

[0365]Two conditionally active antibodies (CAB-scFv-63.9-4 and CAB-scFv-63.9-6) for a drug target antigen X1 were expressed as homodimers with wild type human IgG1 Fc (resulting in bivalent antibodies CAB-scFv-63.9-4-01 and CAB-scFv-63.9-6-01 in FIGS. 2-3), as well as heterodimers in the knob-in-hole system resulting in a monovalent scFv (resulting in monovalent antibodies scFv CAB-scFv-63.9-4-02 and CAB-scFv-63.9-6-02 in FIGS. 2-3).

[0366]The binding affinities of these antibodies to the drug target antigen X1 at pH 6.0 and pH 7.4 were measured by the ELISA assay. As show in FIG. 2, the scFv antibodies showed affinities to drug target antigen X1 at both pH 6.0 and pH 7.4, which were comparable to the full bivalent antibodies. Further, the selectivity of these scFv antibodies at pH 6.0 over pH 7.4 as shown in FIG. 3 was also comparable to the full bivalent antibodies. This example demonstrated that the conditionally active antibodies ...

example 2

scFv Antibodies Against Target Antigen X1 for Constructing CAR-T Cells

[0367]Conditionally active antibodies for the drug target antigen X1 were generated by simultaneously screening for selectivity and affinity, as well as expression level at both pH 6.0 and pH 7.4, in accordance with one embodiment of the present invention. The screening was done in serum using a FLAG tag because there were human antibodies in the serum which might cause false positives for the screening. The screening buffer was a carbonate buffer (krebs buffer with ringer—standard buffer but different from PBS). The generated conditionally active antibodies were found to have a higher affinity to the drug target antigen X1 at pH 6.0 but lower affinity to the same drug target antigen X1 at pH 7.4, both in comparison with the wild-type antibody. Further, these conditionally active antibodies all have high expression levels as shown in Table 2 below, with column “Clone” showing the antibodies and the expression leve...

example 3

CAR-T Cells with a Conditionally Active scFv Antibody Against Target Antigen X1

[0377]A conditionally active scFv antibody against target antigen X1 was used to construct a CAR molecule. T cells were transduced with the CAR molecule such that the T cells expressed the CAR molecule (CAR-T cells). CHO cells expressing target antigen X1 (CHO-63 cells) or regular CHO cells that do not express target antigen X1 (CHO cells) were separately treated with the CAR-T cells. Non-transduced T-cells (without the CAR molecule) were used as a control (FIGS. 8A-8B).

[0378]Referring to FIG. 8A, CHO cells that do not express the target antigen X1 were treated with the CAR-T cells and non-transduced T-cells. There was no significant difference between the two treatments, indicating no cytotoxicity of the CAR-T cells to the CHO cells. Referring to FIG. 8B where CHO cells expressing target antigen X1 (CHO-63) were similarly treated, the CAR-T cells with a conditionally active antibody against target antige...

RELATED APPLICATION DATA

[0001] This application claims benefit of U.S. provisional application No. 62 / 043,067, filed on Aug. 28, 2014, now expired, and international application no. PCT / US15 / 47197, filed on Aug. 27, 2015, and which designates the United States of America, both of which applications are hereby incorporated by reference in their entirety.FIELD OF THE DISCLOSURE

[0002] This disclosure relates to the field of protein evolution. Specifically, this disclosure relates to a method of generating a conditionally active chimeric antigen receptor from a wild type protein. The conditionally active chimeric antigen receptor is reversibly or irreversibly inactivated at a wild type normal physiological condition, but is active at an aberrant condition.BACKGROUND OF THE DISCLOSURE

[0003] There is a considerable body of literature describing the potential for evolving proteins for a variety of characteristics, especially enzymes. For example, enzymes may be evolved to be stabilized for op...

Examples

example 1

Generation of scFv Conditionally Active Antibodies

[0365]Two conditionally active antibodies (CAB-scFv-63.9-4 and CAB-scFv-63.9-6) for a drug target antigen X1 were expressed as homodimers with wild type human IgG1 Fc (resulting in bivalent antibodies CAB-scFv-63.9-4-01 and CAB-scFv-63.9-6-01 in FIGS. 2-3), as well as heterodimers in the knob-in-hole system resulting in a monovalent scFv (resulting in monovalent antibodies scFv CAB-scFv-63.9-4-02 and CAB-scFv-63.9-6-02 in FIGS. 2-3).

[0366]The binding affinities of these antibodies to the drug target antigen X1 at pH 6.0 and pH 7.4 were measured by the ELISA assay. As show in FIG. 2, the scFv antibodies showed affinities to drug target antigen X1 at both pH 6.0 and pH 7.4, which were comparable to the full bivalent antibodies. Further, the selectivity of these scFv antibodies at pH 6.0 over pH 7.4 as shown in FIG. 3 was also comparable to the full bivalent antibodies. This example demonstrated that the conditionally active antibodies ...

example 2

scFv Antibodies Against Target Antigen X1 for Constructing CAR-T Cells

[0367]Conditionally active antibodies for the drug target antigen X1 were generated by simultaneously screening for selectivity and affinity, as well as expression level at both pH 6.0 and pH 7.4, in accordance with one embodiment of the present invention. The screening was done in serum using a FLAG tag because there were human antibodies in the serum which might cause false positives for the screening. The screening buffer was a carbonate buffer (krebs buffer with ringer—standard buffer but different from PBS). The generated conditionally active antibodies were found to have a higher affinity to the drug target antigen X1 at pH 6.0 but lower affinity to the same drug target antigen X1 at pH 7.4, both in comparison with the wild-type antibody. Further, these conditionally active antibodies all have high expression levels as shown in Table 2 below, with column “Clone” showing the antibodies and the expression leve...

example 3

CAR-T Cells with a Conditionally Active scFv Antibody Against Target Antigen X1

[0377]A conditionally active scFv antibody against target antigen X1 was used to construct a CAR molecule. T cells were transduced with the CAR molecule such that the T cells expressed the CAR molecule (CAR-T cells). CHO cells expressing target antigen X1 (CHO-63 cells) or regular CHO cells that do not express target antigen X1 (CHO cells) were separately treated with the CAR-T cells. Non-transduced T-cells (without the CAR molecule) were used as a control (FIGS. 8A-8B).

[0378]Referring to FIG. 8A, CHO cells that do not express the target antigen X1 were treated with the CAR-T cells and non-transduced T-cells. There was no significant difference between the two treatments, indicating no cytotoxicity of the CAR-T cells to the CHO cells. Referring to FIG. 8B where CHO cells expressing target antigen X1 (CHO-63) were similarly treated, the CAR-T cells with a conditionally active antibody against target antige...

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