FIELD

The present disclosure relates to combination therapies useful for the treatment of cancers. Certain embodiments relate to a combination therapy which comprises a HPK1 inhibitor or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising such compounds or salts, in combination with a PD-1 axis binding antagonist. The disclosure also relates to associated methods of treatment, pharmaceutical compositions, and pharmaceutical uses. The methods and compositions are useful for any indication for which the therapeutic is itself useful in the detection, treatment and/or prevention of a disease, disorder, or other condition of a subject.

BACKGROUND

Hematopoietic progenitor kinase 1 (HPK1 ), also known as mitogen activated protein kinase kinase kinase kinase 1 (MAP4K1 ), is a member of the mammalian Ste20- like family of serine/threonine kinases that operates via the JNK and ERK signaling pathways. HPK1 is mainly expressed in hematopoietic organs and cells (e.g., T-cells, B- cells, and dendritic cells), suggesting potential involvement of HPK1 in the regulation of signaling in hematopoietic lineages, including lymphocytes. (Shui, et al, “Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses,” Nature Immunology 2006, 8, 84-91 ).

The capacity of T cells to recognize tumor-associated antigens (including neoantigens and cancer-testis antigens) is limited by HPK1 (Sawasdikosol et al., “A perspective on HPK1 as a novel immuno-oncology drug target,” Elife 2020, 9; Di Bartolo et al., “A novel pathway down-modulating T cell activation involves HPK-1 -dependent recruitment of 14-3-3 proteins on SLP-76,” J. Exp. Med. 2007, 204(3) : 681 -91 ; Shui et al., “Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses,” Nat. Immunol. 2007, 8(1 ):84-91 ). When the T-Cell Receptor (TCR) engages a neo-antigen, the TCR-associated kinase Lek initiates a signaling cascade involving SLP76 that ultimately results in T cell proliferation, cytokine secretion, and/or anti-tumor cytotoxicity. However, Lek also phosphorylates and activates HPK1 which, in turn, phosphorylates and targets for SLP76 degradation (Liou et al., “HPK1 is activated by lymphocyte antigen receptors and negatively regulates AP-1 ,” Immunity, 2000, 12(4) : 399-408; Ling et al., “Involvement of hematopoietic progenitor kinase 1 in T cell receptor signaling,” J. Biol. Chem., 2001 , 276(22) : 18908-14; Sauer et al., “Hematopoietic progenitor kinase 1 associates physically and functionally with the adaptor proteins B cell linker protein and SLP-76 in lymphocytes,” J. Biol. Chem., 2001 , 276 (48) : 45207-16). HPK1 -mediated loss of SLP76 stymies recognition of suboptimal stimuli.

The programmed death 1 (PD-1 ) receptor is expressed by activated T cells, B cells, and myeloid cells. PD-1 has two known ligands, programmed death ligand 1 (PD- L1 ) and programmed death ligand 2 (PD-L2). PD-1 is activated by PD-L1 (also referred to as B7-H1 , B7-4, CD274, and B7-H) and PD-L2 expressed by stromal cells, tumor cells, or both, initiating T-cell death and localized immune suppression (Dong et al., “B7-H1 , a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion,” Nat. Med. 1999; 5:1365-69; Freeman et al., “Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation,” J. Exp. Med. 2000; 192:1027-34). Engagement the PD-1 receptor on T cells by PD-L1 results in suppression of CD28 costimulatory signaling (Kamphorst et al., “Rescue of exhausted CD8 T cells by PD-1 -targeted therapies is CD28-dependent,” Science 2017, 355 (6332): 1423-7; Hui et al., “T cell costimulatory receptor CD28 is a primary target for PD-1 -mediated inhibition,” Science 2017, 355(6332): 1428-33). Conversely, inhibition of this interaction can enhance local T-cell responses and mediate antitumor activity in nonclinical animal models (Iwai Y, et al., “Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade,” Proc. Natl. Acad. Set. USA 2002; 99:12293-97).

PD-L1 is a cell-surface protein and member of the B7 family. PD-L1 is found on almost all types of lymphohematopoietic cells and is expressed at low levels by resting T cells, B cells, macrophages and dendritic cells and is further up regulated by an anti- CD40 antibody for B cells, anti-CD3 antibody for T cells, anti-CD40 antibody, IFNy and granulocyte macrophage colony-stimulating factor (GM-CSF) for macrophages and/or anti-CD40 antibody, IFNy, IL-4, IL-12 and GM-CSF for Dendritic cells (DCs). PD-L1 is also expressed by some non-hemoatopoietic cells and is overexpressed in many cancers, wherein its overexpression is often associated with poor prognosis (Okazaki T et al., PD-1 and PD-1 ligands: from discovery to clinical application, Intern. Immun. 2007 19(7):813) (Thompson R H et al., “Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up,” Cancer Res 2006, 66(7):3381 ). Interestingly, the majority of tumor infiltrating T lymphocytes predominantly express PD- 1 , in contrast to T lymphocytes in normal tissues and peripheral blood. PD-1 on tumor- reactive T cells can contribute to impaired antitumor immune responses (Ahmadzadeh et al., “Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD- 1 and are functionally impaired,” Blood 2009 114(8): 1537). This may be due to exploitation of PD-L1 signaling mediated by PD-L1 expressing tumor cells interacting with PD-1 expressing T cells to result in attenuation of T cell activation and evasion of immune surveillance (Sharpe et al., “The B7-CD28 superfamily,” Nat. Rev. 2002) (Keir ME et al., PD-1 and its ligands in tolerance and immunity, Annu. Rev. Immunol. 2008, 26:677). Therefore, inhibition of the PD-L1 /PD-1 interaction may enhance CD8+ T cell-mediated killing of tumors.

There remains a need for new therapies for the treatment of cancers using a HPK1 inhibitor in combination with a PD-1 axis binding antagonist. The present disclosure is directed toward this need and others. Combination therapies of the present disclosure show greater efficacy than treatment with the individual therapeutic agents alone.

All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.

SUMMARY

This disclosure relates to therapeutic methods, combinations, and pharmaceutical compositions for use in the treatment of cancer. Also provided are combination therapies comprising the compounds of the disclosure, in combination with other therapeutic agents. The present disclosure also provides kits comprising one or more of the compositions of the disclosure.

In one aspect, the disclosure provides a method for treating cancer comprising administering to a subject in need thereof an amount of a HPK1 inhibitor in combination with an amount of a PD-1 axis binding antagonist, wherein the amounts together are effective in treating cancer; wherein the HPK1 inhibitor is a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein:

R ^1a and R ^1b are each independently (Ci-Ce)alkyl, or R ^1a and R ^1b taken together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl that is substituted with 0, 1 , or 2 (Ci-Ce)alkyl;

R ^2a and R ^2b are each independently hydrogen or (Ci-C3)alkyl;

R ^3a and R ^3b are each independently hydrogen or (Ci-C3)alkyl; each R ⁴ is independently (Ci-Ce)alkyl substituted with 0 or 1 halogen or hydroxy; and n is 0, 1 , or 2.

In some preferred embodiments of the methods as described herein, the compound is 4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2 , 1 - c][1 ,2 , 4]triazol-3-y l]pyrid in-2-y l}-6-[(2R)-2-m ethy Ipyrrol idin-1 -y l]-2 , 3-d ihydro-1 H- pyrrolo[3,4-c]pyridin-1 -one (PF-07265028) having the following structure: In some embodiments of the methods as described herein, the PD-1 axis binding antagonist comprises a PD-1 binding antagonist, a PD-L1 binding antagonist, or a PD-L2 binding antagonist.

In some embodiments, the PD-1 axis binding antagonist comprises a PD-1 binding antagonist. In some such embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-LI. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In some such embodiments, the anti-PD-1 antibody is sasanlimab, nivolumab, pembrolizumab, pidilizumab, cemiplimab, tislelizumab, spartalizumab, sintilimab, MEDI- 0680, BGB-108, or AGEN2034 genolimzumab, CBT-502, camrelizumab, or a combination thereof. In one preferred embodiment, the anti-PD-1 antibody is sasanlimab, nivolumab, or pembrolizumab. In another preferred embodiment, the anti-PD-1 antibody is sasanlimab or nivolumab.

In some embodiments of the methods as described herein, the PD-1 axis binding antagonist comprises a PD-L1 binding antagonist. In some embodiments, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.

In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a more specific embodiment, the anti-PD-L1 antibody is BMS-936559, KN035, atezolizumab, durvalumab, avelumab, or a combination thereof.

In one aspect, the disclosure provides a combination comprising a compound of Formula (I) and a PD-1 axis binding antagonist, for use in the treatment of cancer in a subject in need thereof, and a pharmaceutically acceptable carrier.

In one aspect, the disclosure provides a medicament comprising a compound of Formula (I) for use in combination with a PD-1 axis binding antagonist for treating a cancer. In one aspect, the disclosure provides a use of a combination comprising a compound of Formula (I) and a PD-1 axis binding antagonist for the treatment of cancer in a subject in need thereof.

In one aspect, the disclosure provides a kit that comprises a compound of Formula (I), a PD-1 axis binding antagonist, and a package insert, and the package insert comprises instructions for treating a subject for cancer using the medicaments. Such kit can be used, for example, in the methods of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present disclosure will be described herein below with reference to the figures wherein:

FIG. 1 shows the effect of PF-07265028 alone and in combination with an anti- PD1 antibody sasanlimab on the growth of the A375 tumor cells according to certain embodiments of the present disclosure.

FIG. 2 shows the dose response of PF-07265028 alone and in combination with an anti-PD1 antibody sasanlimab on the growth of the A375 tumor cells in a T cell coculture according to certain embodiments of the present disclosure.

FIG. 3 shows the effect of PF-07265028 alone and in combination with an anti- PD1 antibody sasanlimab on the growth of the A375 apoptotic tumor cells in a T cell coculture according to certain embodiments of the present disclosure.

FIG. 4 shows the effect of PF-07265028 alone and in combination with an anti- PD1 antibody sasanlimab on IFNy production in a T cell co-culture with tumor cells according to certain embodiments of the present disclosure.

FIG. 5 shows the effect of PF-07265028 alone and in combination with an anti- PD1 antibody nivolumab on the growth of the A375 tumor cells according to certain embodiments of the present disclosure.

FIG. 6 shows the dose response of PF-07265028 alone and in combination with nivolumab on the growth of the A375 tumor cells in a T cell co-culture according to certain embodiments of the present disclosure.

FIG. 7 shows the effect of PF-07265028 alone and in combination with an anti- PD1 antibody nivolumab on the percent of Ki67-positive CD8 T cells in a A375 tumor cell co-culture according to certain embodiments of the present disclosure. FIG. 8 shows the effect of PF-07265028 alone and in combination with an anti- PD1 antibody nivolumab on the IFNy produced per activated T cell in a A375 tumor cell co-culture according to certain embodiments of the present disclosure.

FIG. 9 shows the effect of PF-07265028 alone and in combination with anti-PD1 antibody nivolumab on the amount of IFNy produced by human donor ‘subject T T cells in co-culture with MDA-MB-231 tumor cells according to certain embodiments of the present disclosure.

FIG. 10 shows the effect of PF-07265028 alone and in combination with anti-PD1 antibody nivolumab on the amount of IFNy produced by human donor ‘subject 2’ T cells in co-culture with MDA-MB-231 tumor cells according to certain embodiments of the present disclosure.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the Examples included herein. It is to be understood that this invention is not limited to specific synthetic methods of making that may of course vary. It is to be also understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

E1 . A method for treating cancer comprising administering to a subject in need thereof an amount of a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein:

R ^1a and R ^1b are each independently (Ci-Ce)alkyl, or R ^1a and R ^1b taken together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl that is substituted with 0, 1 , or 2 (Ci-Ce)alkyl;

R ^2a and R ^2b are each independently hydrogen or (Ci-C4)alkyl;

R ^3a and R ^3b are each independently hydrogen or (Ci-C4)alkyl; each R ⁴ is independently (Ci-Ce)alkyl substituted with 0 or 1 halogen or hydroxy; and n is 0, 1 , or 2; in combination with an amount of a Programmed Death 1 protein (PD-1 ) axis binding antagonist, wherein the amounts together are effective in treating cancer.

E2. The method of embodiment E1 , wherein R ^1a and R ^{1 b} are each independently methyl, ethyl, or isopropyl.

E3. The method of embodiment E1 or E2, wherein R ^{1 a} and R ^{1 b} taken together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl that is substituted with 0 or 1 substituent that is methyl.

E4. The method of any one of embodiments E1 to E3, wherein R ^2a and R ^2b are hydrogen.

E5. The method of any one of embodiments E1 to E4, wherein R ^3a and R ^3b are each independently hydrogen, methyl, or ethyl.

E6. The method of any one of embodiments E1 to E5, wherein R ⁴ is methyl or ethyl.

E7. The method of any one of embodiments E1 to E6, wherein n is 1 .

E8. The method of any one of embodiments E1 to E7, wherein the compound of

Formula (I) is:

4-[1 -aminopropyl]-2-{6-[5-methyl-6,7-dihydro-5H-pyrrolo[2,1 -c][1 ,2,4]triazol-3- y l]pyrid in-2-y l}-6-[2-m ethy Ipyrrol idin-1 -y l]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one;

4-[1 -aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5H-pyrrolo[2,1 -c][1 ,2,4]triazol-3- y l]pyrid in-2-y l}-6-[2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one;

4-[1 -aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5/-/-pyrrolo[2,1-c][1 ,2,4]triazol-3- y l]pyrid in-2-y l}-6-[2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 /-/-pyrrolo[3,4-c]pyrid in-1 -one; 4-[1 -aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5/-/-pyrrolo[2,1-c][1 ,2,4]triazol-3- yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-2,3-dihydro-1/ -/-pyrrolo[3,4-c]pyridin-1-one; or

4-[1 -aminopropyl]-2-{3-[5-methyl-6,7-dihydro-5/-/-pyrrolo[2,1 -c][1 ,2,4]triazol-3- yl]phenyl}-6-[methyl(propan-2-yl)amino]-2,3-dihydro-1 /-/-pyrrolo[3,4-c]pyrid in-1 -one; or a pharmaceutically acceptable salt thereof.

E9. The method of any one of embodiments E1 to E8, wherein the compound of Formula (I) is:

4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2 , 1 - c][1 ,2 ,4]triazol-3-y l]pyrid in-2-y l}-6-[(2R)-2-m ethy Ipyrrol idin-1 -y l]-2 , 3-d ihydro-1 H- pyrrolo[3,4-c]pyridin-1 -one;

4-[(1 R)-1 -aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2,1 - c][1 ,2 ,4]triazol-3-y l]pyrid in-2-yl}-6-[(2R)-2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 H- pyrrolo[3,4-c]pyridin-1 -one;

4-[(1 S)-1 -aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2,1- c][1 ,2 ,4]triazol-3-y l]pyrid in-2-yl}-6-[(2R)-2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 H- pyrrolo[3,4-c]pyridin-1 -one;

4-[(1 R)-1 -aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2,1 - c][1 ,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino] -2,3-dihydro-1 H- pyrrolo[3,4-c]pyridin-1 -one; or

4-[(1 R)-1 -aminopropyl]-2-{3-[(5S)-5-methyl-6,7-dihydro-5H-pyrrolo[2,1 - c][1 ,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]-2,3-d ihydro-1 H-pyrrolo[3,4- c]pyridin-1-one; or a pharmaceutically acceptable salt thereof.

E10. The method of any one of embodiments E1 to E9, wherein the compound of Formula (I) is:

4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2 , 1 - c][1 ,2 ,4]triazol-3-y l]pyrid in-2-yl}-6-[(2R)-2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 /-/- pyrrolo[3,4-c]pyridin-1 -one.

E11 . The method of any one of embodiments E1 to E10, wherein the PD-1 axis binding antagonist comprises a PD-1 antagonist, a PD-L1 antagonist, or a PD-L2 antagonist. E12. The method of embodiment E11 , wherein the PD-1 axis binding antagonist comprises a PD-1 antagonist. E13. The method of embodiment E11 or E12, wherein the PD-1 binding antagonist is an anti-PD-1 antibody.

E14. The method of embodiment E13 wherein the anti-PD-1 antibody is selected from the group consisting of sasanlimab, nivolumab, pembrolizumab, pidilizumab, cemiplimab, tislelizumab, spartalizumab, sintilimab, MEDI-0680, BGB-108, AGEN2034 genolimzumab, CBT-502, camrelizumab, and a combination thereof.

E15. The method of embodiment E14, wherein the anti-PD-1 antibody is sasanlimab. E16. The method of embodiment E14, wherein the anti-PD-1 antibody is nivolumab.

E17. The method of embodiment E14, wherein the anti-PD-1 antibody is pembrolizumab.

E18. The method of embodiment E11 or E12, wherein the PD-1 axis binding antagonist comprises a PD-L1 antagonist.

E19. The method of embodiment E18, wherein the PD-L1 binding antagonist is an anti- PD-L1 antibody.

E20. The method of any one of embodiments E1 to E19, wherein the subject is a human.

E21 . The method of any one of embodiments E1 to E20, wherein the cancer is selected from the group consisting of brain cancer, head/neck cancer, prostate cancer, bladder cancer, lung cancer, breast cancer, ovarian cancer, bone cancer, colorectal cancer, kidney cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, gastroesophageal junction cancer, thyroid cancer, cervical cancer, uterine cancer, and renal cancer.

E22. A combination for use in the treatment of cancer in a subject in need thereof comprising:

(i) a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein:

R ^1a and R ^1b are each independently (Ci-Ce)alkyl, or R ^1a and R ^1b taken together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl that is substituted with 0, 1 , or 2 (Ci-Ce)alkyl;

R ^2a and R ^2b are each independently hydrogen or (Ci-C4)alkyl;

R ^3a and R ^3b are each independently hydrogen or (Ci-C4)alkyl; each R ⁴ is independently (Ci-Ce)alkyl substituted with 0 or 1 halogen or hydroxy; and n is 0, 1 , or 2; and

(ii) a PD-1 axis binding antagonist.

E23. The combination of embodiment E22, wherein the HKP1 inhibit is 4-[(1R)-1 - aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2, 1 -c][1 ,2,4]triazol-3-yl]pyridin- 2-yl}-6-[(2R)-2-methylpyrrolidin-1 -y l]-2 , 3-d ihydro-1 H-py rrolo[3, 4-c]py rid in-1 -one, and the PD-1 axis binding antagonist comprises an anti-PD-1 antibody selected from the group consisting of sasanlimab, nivolumab, pembrolizumab, pidilizumab, cemiplimab, tislelizumab, spartalizumab, sintilimab, MEDI-0680, BGB-108, or AGEN2034 genolimzumab, CBT-502, camrelizumab, and a combination thereof.

E24. The combination of embodiment E22 or E23, wherein the PD-1 axis binding antagonist comprises sasanlimab.

E25. The combination of embodiment E22 or E23, wherein the PD-1 axis binding antagonist comprises nivolumab. E26. The combination of embodiment E22 or E23, wherein the PD-1 axis binding antagonist comprises pembrolizumab.

E27. Use of a combination of any one of embodiments E22 to E26 for the treatment of cancer in a subject in need thereof.

I. Definitions

As used herein, the singular form "a," "an," and "the" include plural references unless indicated otherwise. For example, "a" substituent includes one or more substituents. Where the plural form is used for compounds, salts, and the like, this is taken to mean also a single compound, salt, or the like.

The disclosure described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of' may be replaced with either of the other two terms.

The term “about” when used to modify a numerically defined parameter means that the parameter may vary by as much as 10% above or below the stated numerical value for that parameter.

The term "alkyl" refers to a saturated, monovalent aliphatic hydrocarbon radical including straight chain and branched chain groups having the specified number of carbon atoms. The alkyl substituents disclosed herein may be specified as 1 to 6 carbon atoms (“C1-C6 alkyl”), or 1 to 4 carbon atoms (“C1-C4 alkyl”), and so forth. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl and the like. Alkyl groups may be substituted or unsubstituted. In particular, unless otherwise specified, alkyl groups may be substituted by one or more halo groups, up to the total number of hydrogen atoms present on the alkyl moiety. Thus, C1-C4 alkyl includes halogenated alkyl groups, and in particular fluorinated alkyl groups, having 1 to 4 carbon atoms, e.g., trifluoromethyl or difluoroethyl (i.e., CF3 and -CH2CHF2).

In other embodiments, alkyl is optionally substituted by one or more substituent, for example, by 1 to 3 substituents, independently selected from the group consisting of halo, -OH, C1-C4 alkoxy, -CN, and -NR ^x R ^y . The term “alkoxy” refers to a monovalent -O-alkyl group, wherein the alkyl portion has the specified number of carbon atoms. Alkoxy groups typically contain 1 to 8 carbon atoms (“C1-C8 alkoxy”), or 1 to 6 carbon atoms (“C-i-Ce alkoxy”), or 1 to 4 carbon atoms (“C1-C4 alkoxy”). For example, C1-C4 alkoxy includes methoxy, ethoxy, isopropoxy, tertbutyloxy (i.e., -OCH3, -OCH2CH3, -OCH(CH ₃ ) ₂ , -OC(CH ₃ ) ₃ ), and the like.

The term "heterocycloalkyl" refers to a non-aromatic, saturated or partially unsaturated ring system containing the specified number of ring atoms, including at least one heteroatom selected from N, 0 and S as a ring member. A 5-membered heterocycloalkyl may include tetraphydrofuran (tetrahydrofuranyl), tetrahydrothiophene (tetrahydrothiophenyl), and pyrrolidine (pyrrolidinyl).

As used herein, terms, including, but not limited to, “drug,” “agent,” “component,” “composition," “compound," “substance," “targeted agent," “targeted therapeutic agent," “therapeutic agent,” and “medicament” may be used interchangeably to refer to the small molecule compounds of the present disclosure, e.g., a HPK1 inhibitor, an anti-PD-L1 antibody, an anti-PD-1 antibody, or combinations thereof.

As used herein, terms “therapeutic antibody” and “antibody” may be used interchangeable to refer to an antibody that is used in the treatment of a disease or a disorder. A therapeutic antibody may have various mechanisms of action. A therapeutic antibody may bind and neutralize the normal function of a target associated with an antigen. For example, a monoclonal antibody that blocks the activity of the protein needed for the survival of a cancer cell causes the cell's death. Another therapeutic antibody may bind and activate the normal function of a target associated with an antigen. For example, a monoclonal antibody can bind to a protein on a cell and trigger an apoptosis signal. Yet another monoclonal antibody may bind to a target antigen expressed only on diseased tissue; conjugation of a toxic payload (effective agent), such as a chemotherapeutic or radioactive agent, to the monoclonal antibody can create an agent for specific delivery of the toxic payload to the diseased tissue, reducing harm to healthy tissue. A “biologically functional fragment” of a therapeutic antibody will exhibit at least one if not some or all of the biological functions attributed to the intact antibody, the function comprising at least specific binding to the target antigen. The therapeutic antibody may bind to any protein, including, without limitation, a PD-L1 , a PD-1. Accordingly, therapeutic antibodies include, without limitation, anti-PD- L1 antibodies, anti-PD-1 antibodies, or combinations thereof.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT- 11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; pemetrexed; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1 -TM1 ); eleutherobin; pancratistatin; TLK-286; CDP323, an oral alpha-4 integrin inhibitor; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omegal (e.g., Nicolaou et al., Angew. Chem Inti. Ed. Engl., 33: 183- 186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-Fll); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and imatinib (a 2- phenylaminopyrimidine derivative), as well as other c- it inhibitors; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDIS1 NE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin. Additional examples of chemotherapeutic agents include anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 1 1 7018, onapristone, and toremifene (FARESTON®); anti-progesterones; estrogen receptor down-regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX®); agents that function to suppress or shut down the ovaries, for example, luteinizing hormone-releasing hormone (LHRFI) agonists such as leuprolide acetate (LLIPRON® and ELIGARD®), goserelin acetate, buserelin acetate and tripterelin; antiandrogens such as fiutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole, vorozole (RJVISOR®), letrozole (FEMARA®), and anastrozole (AR I Ml DEX®). In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); as well as troxacitabine (a 1 ,3- dioxolane nucleoside cytosine analog); anti-sense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); an anti-estrogen such as fulvestrant; a Kit inhibitor such as imatinib or EXEL-0862 (a tyrosine kinase inhibitor); EGFR inhibitor such as erlotinib or cetuximab; an anti-VEGF inhibitor such as bevacizumab; arinotecan; rmRH (e.g., ABARELIX®); lapatinib and lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor also known as GW572016); 17AAG (geldanamycin derivative that is a heat shock protein (Hsp) 90 poison), and pharmaceutically acceptable salts, acids or derivatives of any of the above. The term "immunotherapy" refers to the treatment of a subject by a method comprising inducing, enhancing, suppressing, or otherwise modifying an immune response.

The terms “abnormal cell growth” and “hyperproliferative disorder” are used interchangeably in this application. “Abnormal cell growth,” as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). Abnormal cell growth may be benign (not cancerous), or malignant (cancerous).

A “disorder” is any condition that would benefit from treatment with the compounds of the present invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the subject to the disorder in question.

The term “antibody,” as used herein, refers to an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a bispecific antibody, a dual-specific antibody, bifunctional antibody, a trispecific antibody, a multispecific antibody, a bispecific heterodimeric diabody, a bispecific heterodimeric IgG, a labeled antibody, a humanized antibody, a human antibody, and fragments thereof (such as Fab, Fab’, F(ab’)2, Fv), single chain (ScFv) and domain antibodies (including, for example, shark and camelid antibodies), fusion proteins comprising an antibody, any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site, and antibody like binding peptidomimetics (ABiPs). An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG-1 , IgG- 2, lgG-3, lgG-4, lgA1 and lgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and £ isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, e.g., Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Basic and Clinical Immunology, 8th Edition, 1994, page 71 and Chapter s. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes.

"Monoclonal antibody" or “mAb” or “Mab,” as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 1975, 256: 495; or may be made by recombinant DNA methods (e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Making antibody fragments using phage display libraries, Nature 1991 , 352: 624-628 and Marks et al., By-passing immunization: human antibodies from V-gene libraries displayed on phage, J. Mol. Biol. 1991 , 222: 581 -597, for example. See also Presta, Selection, design, and engineering of therapeutic antibodies, J. Allergy Clin. Immunol. 2005,116:731.

"Chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

“Human antibody” refers to an antibody that comprises human immunoglobulin protein sequences only. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” refer to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

"Humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The prefix “hum,” “hu” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.

The phrase "substantially reduced," "substantially different," or “substantially inhibit,” as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

As use herein, the term "specifically binds to" or is "specific for" refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10 percent of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (/.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as lgG-1 , lgG-2 (including lgG2A and lgG2B), lgG-3, or lgG-4 subtypes, IgA (including IgA- 1 and IgA-2), IgE, IgD or IgM. The Ig fusions preferably include the substitution of a domain of a polypeptide or antibody described herein in the place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an lgG-1 molecule. For the production of immunoglobulin fusions, see also US Patent No. 5,428,130 issued June 27, 1995. Immunoadhesin combinations of Ig Fc and ECD of cell surface receptors are sometimes termed soluble receptors.

A "fusion protein" and a "fusion polypeptide" refer to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property. The property may be a biological property, such as activity in vitro or in vivo. The property may also be simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two portions may be linked directly by a single peptide bond or through a peptide linker but are in reading frame with each other.

A "PD-1 oligopeptide," "PD-L1 oligopeptide," or "PD-L2 oligopeptide" is an oligopeptide that binds, preferably specifically, to a PD-1 , PD-L1 or PD-L2 negative costimulatory polypeptide, respectively, including a receptor, ligand or signaling component, respectively, as described herein. Such oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. Such oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,

64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,

87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more.

Such oligopeptides may be identified using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (e.g., U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871 , 4,833,092, 5,223,409, 5,403,484, 5,571 ,689, 5,663,143; PCT Publication Nos. WO 1984/0003506 and WO 1984/0003564; Geysen etal., Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid, Proc. Natl. Acad. Sci. 1984, 81 :3998-4002; Geysen et al., Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein, Proc. Natl. Acad. Sci. 1985, 82:178-182; Geysen et al., A priori delineation of a peptide which mimics a discontinuous antigenic determinant, Synthetic Peptides as Antigens, 1986, 130-149; Geysen, et al., Strategies for epitope analysis using peptide synthesis, J. Immunol. Meth. 1987, 102, 259-274; Schoofs et al., Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution, J. Immunol, 1988, 140:611 -616, Cwirla, S. E. et al., Peptides on phage: a vast library of peptides for identifying ligands., Proc. Natl. Acad. Sci. 1990, 87:6378; Lowman, H.B. et al., Selecting high-affinity binding proteins by monovalent phage display, Biochemistry, 1991 , 30:10832; Clackson, T. et al., Making antibody fragments using phage display libraries, Nature, 1991 , 352: 624; Marks, J. D. et al., By-passing immunization: human antibodies from V-gene libraries displayed on phage, J. Mol. Biol, 1991 , 222:581 ; Kang, et al., Linkage of Recognition and Replication Functions by Assembling Combinatorial Antibody Fab Libraries Along Phage Surfaces, PNAS, 1991 , vol. 88, pp. 4363-4366, and Smith, G. P. Surface presentation of protein epitopes using bacteriophage expression systems, Curr. Opin. Biotechnol. 1991 , 2:668.

An "antagonist” antibody or a "blocking" antibody is one that inhibits or reduces a biological activity of the antigen it binds. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. The anti-PD-L1 antibodies of the invention block the signaling through PD-1 so as to restore a functional response by T-cells (e.g., proliferation, cytokine production, target cell killing) from a dysfunctional state to antigen stimulation.

An "agonist" or “activating antibody” is one that enhances or initiates signaling by the antigen to which it binds. In some embodiments, agonist antibodies cause or activate signaling without the presence of the natural ligand.

The term "dysfunction" in the context of immune dysfunction, refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes the common elements of both exhaustion and/or anergy in which antigen recognition may occur, but the ensuing immune response is ineffective to control infection or tumor growth.

The term "dysfunctional", as used herein, also includes refractory or unresponsive to antigen recognition, specifically, impaired capacity to translate antigen recognition into down-stream T-cell effector functions, such as proliferation, cytokine production and/or target cell killing.

The term "anergy" refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T-cell receptor (e.g., increase in intracellular Ca+2 in the absence of ras-activation). T cell anergy can also result upon stimulation with antigen in the absence of co- stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of co stimulation. The unresponsive state can often be overridden by the presence of lnterleukin-2. Anergic T-cells do not undergo clonal expansion and/or acquire effector functions.

The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunoregulatory cytokines) as well as cell intrinsic negative regulatory (co stimulatory) pathways.

"Enhancing T-cell function" means to induce, cause or stimulate a T-cell to have a sustained or amplified biological function, or renew or reactivate exhausted or dysfunctional T-cells. Examples of enhancing T-cell function include: increased secretion of y-interferon from CD4+ or CD8+ T-cells, increased proliferation, increased survival, increased differentiation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) relative to such levels before the intervention. In some embodiments, the level of enhancement is as least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to one of ordinary skill in the art. As used herein, "metastasis" or “metastatic” is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.

The term “cancer,” “cancerous,” or “malignant” refers to or describe the physiological condition in subjects that is typically characterized by unregulated cell growth. The term “cancer” includes but is not limited to a primary cancer that originates at a specific site in the body, a metastatic cancer that has spread from the place in which it started to other parts of the body, a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a person with a history of previous cancer of a different type from the latter one. Examples of cancer include, but are not limited to, brain cancer, head/neck cancer (including squamous cell carcinoma of the head and neck (SCCHN)), prostate cancer, bladder cancer (including urothelial carcinoma, also known as transitional cell carcinoma (TCC)), lung cancer (including squamous cell carcinoma, small cell lung cancer (SCLC), and non-small cell lung cancer (NSCLC)), breast cancer, ovarian cancer, bone cancer, colorectal cancer, kidney cancer, liver cancer (including hepatocellular carcinoma (HCC)), pancreatic cancer, esophageal cancer (including squamous cell carcinoma (SCC)), gastric cancer, gastroesophageal junction cancer, thyroid cancer, cervical cancer, uterine cancer, and/or renal cancer. In some embodiments, the cancer is melanoma.

The term “subject” or “a subject in need thereof” refers to a human or non-human subject including veterinary subjects such as, cows, sheep, cats, dogs, horses, primates, rabbits, and rodents (e.g., mice and rats) for which therapy is desired. In one embodiment, the subject is a human and may be referred to as a patient or individual, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from cancer. Those skilled in the medical art are readily able to identify individual patients who are afflicted with cancer.

The term “treat” or “treating” a cancer, as used herein means to administer a combination therapy according to the present disclosure to a subject having cancer, or diagnosed with cancer, to achieve at least one positive therapeutic effect, such as, for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organize, or reduced rate of tumor metastases or tumor growth, reversing, stopping, controlling, slowing, interrupting, arresting, alleviating, and/or inhibiting the progression or seventy of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. Within the meaning of the present disclosure, the term “treat” or “treating” also denotes, to arrest, delay the onset (i.e. , the period prior to clinical manifestation of a disease or symptom of a disease) and/or reduce the risk of developing or worsening a symptom of a disease.

As used herein, “in combination with,” “combined administration,” or “coadministration” refers to administration of one agent in addition to at least one other agent, where the administration of one agent can be before, during, or after administration of at least one other agent to the individual. In some embodiments, the co-administration of two or more agents can be useful for treating individuals suffering from cancer who have primary or acquired resistance to ongoing therapies. The combination therapy provided herein may be useful for improving the efficacy and/or reducing the side effects of cancer therapies for individuals who do respond to such therapies.

As used herein, the term “simultaneously,” "simultaneous administration,” "administered simultaneously,” “concurrently,” or "concurrent administration,” means that the agents are administered at the same point in time either in separate dosage forms or as part of the same dosage form, or immediately following one another, but that the agents can be administered in any order. For example, in the latter case, the two or more agents are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the agents are administered at the same point in time.

The agents of the present disclosure can be administered completely separately or in the form of one or more separate compositions. For example, the agents may be given separately at different times during the course of therapy (in a chronologically staggered manner, especially a sequence-specific manner) in such time intervals that the combination therapy is effective in treating cancer.

As used herein, the term “sequential,” “sequentially,” “administered sequentially,” or “sequential administration” refers to the administration of each agent of the combination therapy of the invention, either alone or in a medicament, one after the other, wherein each agent can be administered in any order. Sequential administration may be particularly useful when the therapeutic agents in the combination therapy are in different dosage forms, for example, one agent is a tablet and another agent is a sterile liquid, and/or the agents are administered according to different dosing schedules, for example, one agent is administered daily, and the second agent is administered less frequently such as weekly.

The term “single formulation,” as used herein, refers to a single carrier or vehicle formulated to deliver effective amounts of both therapeutic agents to a subject. The single vehicle is designed to deliver an effective amount of each of the agents, along with any pharmaceutically acceptable carriers or excipients. In some embodiments, the vehicle is a tablet, capsule, pill, or a patch. In other embodiments, the vehicle is a solution or a suspension.

The term “unit dose” is used herein to mean simultaneous administration of both agents together, in one dosage form, to the subject being treated. In some embodiments, the unit dose is a single formulation. In certain embodiments, the unit dose includes one or more vehicles such that each vehicle includes an effective amount of at least one of the agents along with pharmaceutically acceptable carriers and excipients. In some embodiments, the unit dose is one or more tablets, capsules, pills, or patches administered to the subject at the same time.

An “oral dosage form” includes a unit dosage form prescribed or intended for oral administration.

The term “advanced,” as used herein, as it relates to breast cancer, includes locally advanced (non-metastatic) disease and metastatic disease.

The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable” carriers or excipients (vehicles, additives) are those which can reasonably be administered to a subject to provide an effective dose of the active ingredient employed.

The combinations provided herein may be formulated by a variety of methods apparent to those of skill in the art of pharmaceutical formulation. The various release properties described above may be achieved in a variety of different ways. Suitable formulations include, for example, tablets, capsules, press coat formulations, and other easily administered formulations.

A "package insert" refers to instructions customarily included in commercial packages of medicaments that contain information about the indications customarily included in commercial packages of medicaments that contain information about the indications, usage, dosage, administration, contraindications, other medicaments to be combined with the packaged product, and/or warnings concerning the use of such medicaments, etc.

An “effective dosage,” “effective amount,” "therapeutically effective amount," or “therapeutically effective dosage” of a drug, agent, component, composition, compound, substance, targeted agent, targeted therapeutic agent, therapeutic antibody, therapeutic agent, medicament or pharmaceutical composition is an amount to affect any one or more beneficial or desired, including biochemical, histological and/or behavioral symptoms-of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.

For therapeutic use, a therapeutically effective amount refers to that amount of a drug, agent, component, composition, compound, substance, targeted agent, targeted therapeutic agent, therapeutic antibody, therapeutic agent, medicament or pharmaceutical composition being administered which will relieve to some extent one or more of the symptoms of the disorder being treated such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival. In reference to the treatment of cancer, a therapeutically effective amount refers to that amount of a drug, agent, component, composition, compound, substance, targeted agent, targeted therapeutic agent, therapeutic antibody, therapeutic agent, medicament or pharmaceutical composition which is effective to achieve one or more of the following results following the administration of one or more therapies: (1 ) reducing the size of the tumor, (2) reducing the number of cancer cells, (3) inhibiting (i.e., slowing to some extent, preferably stopping) cancer cell infiltration into peripheral organs, (4) inhibiting (i.e., slowing to some extent, preferably stopping) tumor metastasis, (5) inhibiting (i.e., slowing to some extent, preferably stopping) tumor growth or tumor invasiveness, (6) relieving (i.e., to some extent, preferably eliminating) one or more signs or symptoms associated with the cancer, (7) decreasing the dose of other medications required to treat the disease, (8) enhancing the effect of another medication, (9) delaying the progression of the disease, (10) improving or increasing the disease-free, relapse-free, progression- free, and/or overall survival, duration, or rate, (11 ) increasing the response rate, the durability of response, or number of patients who respond or are in remission, (12) decreasing the hospitalization rate, (13) decreasing the hospitalization lengths, (14) the size of the tumor is maintained and does not increase or increases by less than 10%, preferably less than 5%, preferably less than 4%, preferably less than 2%; (12) an increase in the number of patients in remission, (15) increasing the length or duration of remission, (16) decreasing the recurrence rate of cancer; (15) decreasing the time to recurrence of cancer, and (17) an amelioration of cancer related symptoms and/or quality of life.

An effective amount can be administered in one or more administrations. For the purposes of this disclosure, an effective amount is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in conjunction with another drug, agent, component, composition, compound, substance, targeted agent, targeted therapeutic agent, therapeutic antibody, therapeutic agent, medicament or pharmaceutical composition.

An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of drug, compound, and/or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.

As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

A therapeutic amount may also refer to a dosage of a drug that has been approved for use by a regulatory agency. A "subtherapeutic amount," as used herein, refers to a dosage of a drug that is significantly lower than the approved dosage.

The terms “treatment regimen,” “dosing protocol” and “dosing regimen” are used interchangeably to refer to the dose and timing of administration of each therapeutic agent in a combination of the invention.

The term "ameliorating,” or "amelioration,” with reference to a disease, disorder or condition, refers to any observable beneficial effect of the treatment. Treatment need not be absolute to be beneficial to the subject. For example, ameliorating means a lessening or improvement of one or more symptoms of a disease, disorder or condition as compared to not administering a therapeutic agent of a method or regimen of the invention. Ameliorating also includes shortening or reduction in duration of a symptom.

The term "biosimilar" refers to a biological product that is highly similar to an FDA- approved biological product (reference product) and has no clinically meaningful differences in terms of pharmacokinetics, safety and efficacy from the reference product.

The term "bioequivalent" refers to a biological product that is pharmaceutically 5 equivalent and has a similar bioavailability to an FDA-approved biological product (reference product). For example, according to the FDA the term bioequivalence is defined as "the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administered at the same molar dose under similar conditions 10 in an appropriately designed study" (United States Food and Drug Administration, "Guidance for Industry: Bioavailability and Bioequicalence Studies for Orally Administered Drug Products - General Considerations," 2003, Center for Drug Evaluation and Research).

“Tumor” as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.

“Tumor burden” also referred to as a “tumor load’, refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of tumor(s), throughout the body, including lymph nodes and bone marrow. Tumor burden can be determined by a variety of methods known in the art, such as, e.g., using calipers, or while in the body using imaging techniques, e.g., ultrasound, bone scan, computed tomography (CT), or magnetic resonance imaging (MRI) scans.

The term “additive” is used to mean that the result of the combination of two or more agents is no greater than the sum of each agent individually. In one embodiment, the combination of agents described herein displays a synergistic effect. The term “synergy” or “synergistic” are used to mean that the result of the combination of two or more agents is greater than the sum of each agent individually. This improvement in the disease, condition or disorder being treated is a “synergistic” effect. A “synergistic amount” is an amount of the combination of the two or more agents that results in a synergistic effect, as “synergistic” is defined herein. A “synergistic combination” refers to a combination of agents which produces a synergistic effect in vivo, or alternatively in vitro as measured according to the methods described herein.

Determining a synergistic interaction between two or more agents, the optimum range for the effect and absolute dose ranges of each agent for the effect may be definitively measured by administration of the agents over different dose ranges, and/or dose ratios to subjects in need of treatment. However, the observation of synergy in in vitro models or in vivo models can be predictive of the effect in humans and other species and in vitro models or in vivo models exist, as described herein, to measure a synergistic effect. The results of such studies can also be used to predict effective dose and plasma concentration ratio ranges and the absolute doses and plasma concentrations required in humans and other species such as by the application of pharmacokinetic and I or pharmacodynamics methods.

A “nonstandard clinical dosing regimen,” as used herein, refers to a regimen for administering a substance, agent, compound or composition, which is different to the amount, dose or schedule typically used for that substance, agent, compound or composition in a clinical setting. A “non-standard clinical dosing regimen,” includes a “non-standard clinical dose” or a “nonstandard dosing schedule”.

The term "pharmaceutically acceptable salt," as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Some embodiments also relate to the pharmaceutically acceptable acid addition salts of the compounds described herein. Suitable acid addition salts are formed from acids which form non-toxic salts. Non-limiting examples of suitable acid addition salts, i.e., salts containing pharmacologically acceptable anions, include, but are not limited to, the acetate, acid citrate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, bitartrate, borate, camsylate, citrate, cyclamate, edisylate, esylate, ethanesulfonate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobrom ide/brom ide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, p-toluenesulfonate, trifluoroacetate and xinofoate salts.

Additional embodiments relate to base addition salts of the compounds described herein. Suitable base addition salts are formed from bases which form non-toxic salts. Non-limiting examples of suitable base salts include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

The compounds described herein that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds described herein are those that form non-toxic acid addition salts, e.g., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1 ,T-methylene-bis-(2-hydroxy- 3-naphthoate)] salts. The compounds described herein that include a basic moiety, such as an amino group, may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.

The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of those compounds described herein that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.

For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Methods for making pharmaceutically acceptable salts of compounds described herein are known to one of skill in the art.

"Carriers," as used herein, include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The compounds described herein may also exist in unsolvated and solvated forms. Accordingly, some embodiments relate to the hydrates and solvates of the compounds described herein. The term “solvate,” as used herein, describes a molecular complex comprising a compound described herein and one or more pharmaceutically acceptable solvent molecules, for example, water (hydrate) and ethanol.

Compounds described herein containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where a compound described herein contains an alkenyl or alkenylene group, geometric cis/trans (or Z/E) isomers are possible. Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism (‘tautomerism’) can occur. This can take the form of proton tautomerism in compounds described herein containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. A single compound may exhibit more than one type of isomerism.

The compounds of the embodiments described herein include all stereoisomers (e.g., cis and trans isomers) and all optical isomers of compounds described herein (e.g., R and S enantiomers), as well as racemic, diastereomeric and other mixtures of such isomers. While all stereoisomers are encompassed within the scope of our claims, one skilled in the art will recognize that particular stereoisomers may be preferred.

In some embodiments, the compounds described herein can exist in several tautomeric forms, including the enol and imine form, and the keto and enamine form and geometric isomers and mixtures thereof. All such tautomeric forms are included within the scope of the present embodiments. Tautomers exist as mixtures of a tautomeric set in solution. In solid form, usually one tautomer predominates. Even though one tautomer may be described, the present embodiments include all tautomers of the present compounds.

Included within the scope of the present embodiments are all stereoisomers, geometric isomers and tautomeric forms of the compounds described herein, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Also included are acid addition or base salts wherein the counterion is optically active, for example, d-lactate or l-lysine, or racemic, for example, dl-tartrate or dl-arginine. Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallization.

Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC).

Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where a compound described herein contains an acidic or basic moiety, a base or acid such as 1- phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.

Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the invention. The materials, methods, and examples are illustrative only and not intended to be limiting.

II. HPK1 Inhibitors

Embodiments of the present invention comprise a compound of Formula I: or a pharmaceutically acceptable salt thereof, wherein: R ^1a and R ^1b are each independently (Ci-Ce)alkyl, or R ^1a and R ^1b taken together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl that is substituted with 0, 1 , or 2 (Ci-Ce)alkyl;

R ^2a and R ^2b are each independently hydrogen or (Ci-C4)alkyl;

R ^3a and R ^3b are each independently hydrogen or (Ci-C4)alkyl; each R ⁴ is independently (Ci-Ce)alkyl substituted with 0 or 1 halogen or hydroxy; and n is 0, 1 , or 2.

In certain embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein R ^1a and R ^1b are each independently methyl, ethyl, isopropyl, and wherein R ^2a , R ^2b , R ^3a , R ^3b , R ⁴ , and n are as defined in any of the herein described embodiments. In some embodiments, one of R ^1a and R ^1b is methyl. In some embodiments, one of R ^1a and R ^1b is ethyl. In some embodiments, one of R ^1a and R ^{1 b} is isopropyl. In some embodiments, both R ^{1 a} and R ^1b are methyl. In some embodiments, one of R ^{1 a} and R ^1b is methyl, and the other one of R ^{1 a} and R ^1b is ethyl. In some embodiments, one of R ^{1 a} and R ^1b is methyl, and the other one of R ^{1 a} and R ^1b is isopropyl.

In certain embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein R ^{1 a} and R ^{1 b} taken together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl that is substituted with 0, 1 , or 2 (Ci-Ce)alkyl, and wherein R ^2a , R ^2b , R ^3a , R ^3b , R ⁴ , and n are as defined in any of the herein described embodiments. In some embodiments, R ^{1 a} and R ^{1 b} taken together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl that is substituted with 0 or 1 methyl.

In certain embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein R ^2a and R ^2b are both hydrogen, and wherein R ^1a , R ^{1 b} , R ^3a , R ^3b and R ⁴ are as defined in any of the herein described embodiments. In certain embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein one of R ^2a and R ^2b is hydrogen and the other one of R ^2a and R ^2b is methyl, and wherein R ^1a , R ^1b , R ^3a , R ^3b , R ⁴ , and n are as defined in any of the herein described embodiments.

In certain embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein R ^3a and R ^3b are each independently hydrogen, methyl, or ethyl, and wherein R ^1a , R ^{1 b} , R ^2a , R ^2b , R ⁴ , and n are as defined in any of the herein described embodiments. In some embodiments, one of R ^3a and R ^3b is hydrogen. In some embodiments, one of R ^3a and R ^3b is methyl. In some embodiments, one of R ^3a and R ^3b is ethyl. In some embodiments, one of R ^3a and R ^3b is hydrogen and the other one of R ^3a and R ^3b is methyl. In some embodiments, one of R ^3a and R ^3b is hydrogen and the other one of R ^3a and R ^3b is ethyl. In some embodiments, both of R ^3a and R ^3b are methyl.

In certain embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein R ⁴ is methyl or ethyl, n is 1 , and wherein R ^{1 a} , R ^{1 b} , R ^2a , R ^2b , R ^3a , and R ^3b are as defined in any of the herein described embodiments. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl. In some embodiments, R ⁴ is methyl substituted with one halogen. In some embodiments, R ⁴ is methyl substituted with one hydroxy. In some embodiments, R ⁴ is attached to the carbon adjacent to the N atom.

In certain embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein each R ⁴ is methyl or ethyl, n is 2, and wherein R ^1a , R ^{1 b} , R ^2a , R ^2b , R ^3a , and R ^3b are as defined in any of the herein described embodiments. In some embodiments, both R ⁴ are methyl.

In certain embodiments, the disclosure provides a compound of Formula II: or a pharmaceutically acceptable salt thereof, wherein R ^2a , R ^2b , R ^3a , and R ⁴ are as defined in any of the herein described embodiments. In some embodiments, the disclosure provides a compound having the absolute stereochemistry with regard to the orientation of R ² , R ^3a , and R ^3b , when R ^3a is different from R ^3b , as shown in Formula Ila: or a pharmaceutically acceptable salt thereof, wherein R ^2a , R ^2b , R ^3a , and R ⁴ are as defined in any of the herein described embodiments.

In some embodiments, the disclosure provides a compound of Formula I, II, Ila, or a pharmaceutically acceptable salt thereof, wherein R ^2a and R ^2b are both hydrogen, R ^3a is hydrogen, R ^3b is methyl or ethyl, and R ⁴ is methyl.

In some embodiments, the disclosure provides a HPK1 inhibitor, wherein the HPK1 inhibitor is a compound that is 4-[1 -aminopropyl]-2-{6-[5-methyl-6,7-dihydro-5H- pyrrolo[2, 1 -c][1 , 2 , 4]triazol-3-y l]py rid in-2-y l}-6-[2-m ethy Ipy rrol idin-1 -y l]-2 , 3-d ihydro-1 H- pyrrolo[3,4-c]pyridin-1 -one;

4-[1 -aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5H-pyrrolo[2,1 -c][1 ,2,4]triazol-3- y l]pyrid in-2-y l}-6-[2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one;

4-[1 -aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5/-/-pyrrolo[2,1-c][1 ,2,4]triazol-3- y l]pyrid in-2-y l}-6-[2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 /-/-pyrrolo[3,4-c]pyrid in-1 -one;

4-[1 -aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5/-/-pyrrolo[2,1-c][1 ,2,4]triazol-3- yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-2,3-dihydro-1/ -/-pyrrolo[3,4-c]pyridin-1-one; or 4-[1 -aminopropyl]-2-{3-[5-methyl-6,7-dihydro-5/-/-pyrrolo[2,1 -c][1 ,2,4]triazol-3- yl]phenyl}-6-[methyl(propan-2-yl)amino]-2,3-dihydro-1 /-/-pyrrolo[3,4-c]pyrid in-1 -one; or a pharmaceutically acceptable salt thereof.

For example, the HPK1 inhibitor of the disclosure is a compound that is 4-[(1R)-1- aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2, 1 -c][1 ,2,4]triazol-3-yl]pyridin- 2-yl}-6-[(2R)-2-methylpyrrolidin-1 -y l]-2 , 3-d ihydro-1 H-py rrolo[3, 4-c]py rid in-1 -one;

4-[( 1 R)-1 -aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2, 1 - c][1 ,2 ,4]triazol-3-y l]pyrid in-2-y l}-6-[(2R)-2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 /-/- pyrrolo[3,4-c]pyridin-1 -one;

4-[( 1 S)-1 -aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2, 1 - c][1 ,2 ,4]triazol-3-y l]pyrid in-2-yl}-6-[(2R)-2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 /-/- pyrrolo[3,4-c]pyridin-1 -one;

4-[( 1 R)-1 -aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2, 1 - c][1 ,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino] -2,3-dihydro-1/-/- pyrrolo[3,4-c]pyridin-1 -one; or

4-[(1 R)-1 -aminopropyl]-2-{3-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2 , 1 - c][1 ,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]-2,3-d ihydro-1/-/-pyrrolo[3,4- c]pyridin-1-one; or a pharmaceutically acceptable salt thereof.

Preferably, the HPK1 inhibitor of the disclosure is 4-[(1 R)-1 -aminopropyl]-2-{6- [(5S)-5-methyl-6,7-dihydro-5H-pyrrolo[2, 1 -c][1 , 2 ,4]triazol-3-y l]py ridi n-2-y l}-6-[(2R)-2- methylpyrrolidin-1 -yl]-2,3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin-1 -one; or a pharmaceutically acceptable salt thereof.

III. PD-1 Axis Binding Antagonists

Embodiments of the present disclosure comprise a PD-1 axis binding antagonist.

As used herein, the term “PD-1 axis binding antagonist” or “PD-1 axis antagonist” refers to a molecule that inhibits the interaction of a PD-1 axis binding partner (e.g., PD- 1 , PD-L1 , PD-L2) with either one or more of its binding partners, for example so as to overcome or partially overcome T-cell dysfunction resulting from signaling on the PD-1 signaling axis — with a result being to restore, partially restore or enhance T-cell function (e.g., proliferation, cytokine production, target cell killing, survival). As used herein, a PD- 1 axis binding antagonist includes one or more of (i) a PD-1 antagonist, (ii) a PD-L1 antagonist, and/or (iii) a PD-L2 antagonist. i) PD-1 antagonist

The term “PD-1 antagonist,” as used herein, refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 , PD-L2. In some embodiments, the PD-1 antagonist is a molecule that inhibits the binding of PD-1 to its binding partners. In a specific aspect, the PD-1 antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. In specific embodiments, the PD-1 antagonist inhibits the binding of PD-1 to PD-L1. In another embodiment, the PD-1 antagonist inhibits the binding of PD-1 to PD-L2. In a further embodiment, the PD-1 antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. For example, PD-1 antagonists include anti- PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In some embodiments, a PD-1 antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as to render a dysfunctional T-cell less non-dysfunctional. In some embodiments, the PD-1 antagonist is an anti-PD-1 antibody (aPD-1 ).

In some embodiments, the PD-1 antagonist blocks binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T cell, B cell or NKT cell) and preferably also blocks binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1 , PD1 , CD279 and SLEB2 for PD-1 ; PDCD1 L1 , PDL1 , B7H1 , B7-4, CD274 and B7-H for PD-L1 ; and PDCD1 L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In any of the treatment method, medicaments and uses of the present invention in which a human individual is being treated, the PD-1 antagonist may block binding of human PD-L1 to human PD-1 , and block binding of both human PD-L1 and PD-L2 to human PD-1 .

PD-1 antagonists useful in any of the treatment methods, medicaments and uses of the present disclosure include a monoclonal antibody (mAb), or antigen binding fragment thereof, which specifically binds to PD-1 or PD-L1 , and preferably specifically binds to human PD-1 or human PD-L1 . The mAb may be a human antibody, a humanized antibody or a chimeric antibody, and may include a human constant region. In some embodiments the human constant region is selected from the group consisting of lgG1 , lgG2, lgG3 and lgG4 constant regions, and in some embodiments, the human constant region is an lgG1 or lgG4 constant region. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv and Fv fragments.

Examples of mAbs that bind to human PD-1 , and useful in the treatment method, medicaments and uses of the present disclosure, are described in U.S. Patent Nos. 7,488,802, 7,521 ,051 , 8,008,449, 8,354,509, 8,168,757, PCT Patent Publication Nos. WO 2004/004771 , WO 2004/072286, WO 2004/056875, and US Patent Publication No. 2011/0271358. Specific anti-human PD-1 mAbs useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present disclosure include: sasanlimab (RN888, PF-06801591 , Pfizer Inc.), nivolumab (OPDIVO®, ONO-4538, B MS -936558, MDX1106, Bristol-Myers Squibb Company), pembrolizumab (KEYTRUDA®, MK-3475, lambrolizumab, Merck & Co., Inc.), pidilizumab (CT-011 ), cemiplimab (LIBTAYO®, REGN2810, Regeneron Pharmaceuticals, Inc.), tislelizumab (BGB-A317, BeiGene Ltd. /Celgene Corporation), spartalizumab (PDR001 , Novartis AG), sintilimab (IBI308, Innovent Biologies, Inc.), MEDI-0680 (AMP-514, AstraZeneca PLC), BGB-108, or AGEN2034 (Agenus Inc.), genolimzumab (CBT-501 , CBT Pharmaceuticals), CBT-502 (CBT Pharmaceuticals), camrelizumab (SHR-1210, Incyte Corporation), or a combination thereof.

In certain embodiments, the PD-1 antagonist is an anti-PD-1 antibody.

Preferably, the anti-PD-1 antibody is pembrolizumab, nivolumab, or sasanlimab. In some preferred embodiments, the anti-PD-1 antibody is nivolumab. In some preferred embodiments, the anti-PD-1 antibody is pembrolizumab. In some preferred embodiments, the anti-PD-1 antibody is sasanlimab. Sasanlimab is a humanized, immunoglobulin G4 (lgG4) monoclonal antibody (mAb) that binds to the PD-1 receptor. By blocking its interaction with PD-L1 and PD-L2, PD-1 pathway-mediated inhibition of the immune response is released, leading to an anti-tumor immune response. Clinical anti-tumor activity with sasanlimab has been seen in a panel of anti-PD1 sensitive solid tumor types including non-small cell lung cancer and urothelial carcinoma. Sasanlimab is described, for example, in US Patent No. US 10,155,037, which is hereby incorporated for all purposes.

Additional exemplary PD-1 antagonists include those described in U.S. Patent Application Publication 20130280265, U.S. Patent Application Publication 20130237580, U.S. Patent Application Publication 20130230514, U.S. Patent Application Publication 20130109843, U.S. Patent Application Publication 20130108651 , U.S. Patent Application Publication 20130017199, U.S. Patent Application Publication 20120251537, U.S. Patent

Application Publication 20110271358, European Patent EP2170959B1 , in PCT

Publication No. WO 2011/066342, PCT Publication No. WO 2015/035606, PCT

Publication No. WO 2015/085847, PCT Publication No. WO 2015/112800, PCT

Publication No. WO 2015/112900, PCT Publication No. WO 2016/092419, PCT

Publication No. WO 2017/017623, PCT Publication No. WO 2017/024465, PCT

Publication No. WO 2017/054646, PCT Publication No. WO 2017/071625, PCT

Publication No. WO 2017/019846, PCT Publication No. WO 2017/132827, PCT

Publication No. WO 2017/214092, PCT Publication No. WO 2018/013017, PCT

Publication No. WO 2018/053106, PCT Publication No. WO 2018/055503, PCT

Publication No. WO 2018/053709, PCT Publication No. WO 2018/068336, and PCT

Publication No. WO 2018/072743, the entire disclosures of which are incorporated herein by reference. Other exemplary PD-1 antagonists are described in Curran et al., PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors, PNAS, 2010, 107, 4275; Topalian et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer, New Engl. J. Med. 2012, 366, 2443; Brahmer et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer, New Engl. J. Med. 2012, 366, 2455; Dolan et al., PD-1 pathway inhibitors: changing the landscape of cancer immunotherapy, Cancer Control 2014, 21 , 3; and Sunshine et al., Pd-1/Pd-L1 Inhibitors, Curr. Opin. in Pharmacol. 2015, 23. ii) PD-L1 antagonist

The term “PD-L1 antagonist,” as used herein, refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L1 with either one or more of its binding partners, such as PD-1 , B7-1. In some embodiments, the PD-1 axis antagonist comprises a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist inhibits the binding of PD-L1 to its binding partners. In a specific aspect, the PD-L1 antagonist inhibits the binding of PD-L1 to PD-1. In another specific aspect, the PD-L1 antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In another specific aspect, the PD-L1 antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.

In some embodiments, the PD-L1 antagonists include anti-PD-L1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 , and/or B7-1 . In some embodiments, a PD-L1 antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L1 so as render a dysfunctional T-cell less non-dysfunctional. In some embodiments, a PD-L1 antagonist is an anti-PD-L1 antibody (aPD-L1 ). In some embodiments, the PD-L1 antibody is a biosimilar or bioequivalent thereof.

In some embodiments, the PD-1 antagonist is an anti-PD-1 antibody.

In some embodiments, the anti-PD-1 antibody useful for this disclosure include

BMS-936559 (Bristol-Myers Squibb Company), KN035 (Jiangsu Alphamab biopharmaceuticals Co., Ltd.), atezolizumab (TECENTRIQ®, MPDL3280A, Roche Holding AG), durvalumab (IMFINZI®, AstraZeneca PLC), and/or avelumab (BAVENCIO®, Merck & Co., Inc. and Pfizer, Inc.).

Additional exemplary PD-L1 antagonists include those described in U.S. Patent

Application Publication 20090055944, U.S. Patent Application Publication 20100203056, U.S. Patent Application Publication 20120039906, U.S. Patent Application Publication 20130045202, U.S. Patent Application Publication 20130309250, U.S. Patent Application Publication US20130034559, U.S. Patent Application Publication US20150282460, U.S.

Patent Application Publication 20160108123, PCT Publication No. WO 2011/066389,

PCT Publication No. WO 2016/000619, PCT Publication No. WO 2016/094273, PCT

Publication No. WO 2016/061142, PCT Publication No. WO 2016/149201 , PCT

Publication No. WO 2016/149350, PCT Publication No. WO 2016/179576, PCT

Publication No. WO 2017/020801 , PCT Publication No. WO 2017/103147, PCT

Publication No. WO 2017/112741 , PCT Publication No. WO 2017/205213, PCT Publication No. WO 2017/054646, PCT Publication No. WO 2017/084495, PCT

Publication No. WO 2017/161976, PCT Publication No. WO 2018/005682, PCT

Publication No. WO 2018/053106, PCT Publication No. WO 2018/085469, PCT

Publication No. WO 2018/111890, and PCT Publication No. WO 2018/106529, the entire disclosures of which are incorporated herein by reference. Other exemplary PD-L1 binding antagonists are described in Sunshine et al., 2015. iii) PD-L2 antagonist

The term “PD-L2 antagonist,” as used herein, refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners. In a specific aspect, the PD-L2 antagonist inhibits binding of PD- L2 to PD-1. In some embodiments, the PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1 . In some embodiments, a PD-L2 antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T-cell less non-dysfunctional. In some embodiments, a PD-L2 antagonist is a PD-L2 immunoadhesin.

IV. METHODS, USES AND MEDICAMENTS

General Methods

Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Edition, 2001 3rd Edition) Molecular Cloning, A Laboratory Manual; Sambrook and Russell Molecular Cloning, 3rd ed., 2001 ; Wu, Recombinant DNA, Vol. 217. Standard methods also appear in Ausbel, et al., Current Protocols in Molecular Biology, Vols.1 -4, 2001 , which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1 ), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4). Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al., Current Protocols in Protein Science, Vol. 1 , 2000, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins are described (e.g., Coligan, et al., Current Protocols in Protein Science, Vol. 2, 2000; Ausubel, et al., Current Protocols in Molecular Biology, Vol. 3, 2001 , pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. Products for Life Science Research, 2001 , pp. 45-89; Amersham Pharmacia Biotech (2001 ) BioDirectory, pp. 384- 391 ). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al., Current Protocols in Immunology, Vol. 1 , 2001 ; Harlow and Lane, Using Antibodies, 1999). Standard techniques for characterizing ligand/receptor interactions are available (e.g., Coligan, et al., Current Protocols in Immunology, Vol. 4, 2001 ).

Monoclonal, polyclonal, and humanized antibodies can be prepared (e.g., Sheperd and Dean (eds.) Monoclonal Antibodies, 2000; Kontermann and Dubel (eds.) Antibody Engineering, 2001 ; Harlow and Lane, Antibodies A Laboratory Manual, 1988, pp. 139-243; Carpenter, et al., Non-Fc receptor-binding humanized anti-CD3 antibodies induce apoptosis of activated human T cells, J. Immunol. 2000, 165:6205; He, et al., Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin,J. Immunol. 1998, 160:1029; Tang et al., Use of a peptide mimotope to guide the humanization of MRK-16, an anti-P-glycoprotein monoclonal antibody , J. Biol. Chem. 1999, 274:27371 -27378; Baca et al., Antibody humanization using monovalent phage display, J. Biol. Chem. 1997, 272:10678-10684; Chothia et al., Conformations of immunoglobulin hypervariable regions, Nature 1989, 342:877-883; Foote and Winter Antibody framework residues affecting the conformation of the hypervariable loops, J. Mol. Biol. 1992, 224:487-499; U.S. Patent No. 6,329,511 ).

An alternative to humanization is to use human antibody libraries displayed on phage or human antibody libraries in transgenic mice (Vaughan et al., Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library, Nature Biotechnol. 1996, 14:309-314; Barbas , Synthetic human antibodies, Nature Medicine 1995, 1 :837-839; Mendez et al., Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice, Nature Genetics 1997, 15:146-156; Hoogenboom and Chames, Natural and designer binding sites made by phage display technology, Immunol. Today 2000, 21 :371 -377; Barbas et al., Phage Display: A Laboratory Manual, 2001 ; Kay et al., Phage Display of Peptides and Proteins: A Laboratory Manual, 1996; de Bruin et al., Selection of high-affinity phage antibodies from phage display libraries, Nature Biotechnol. 1999, 17:397-399).

Purification of antigen is not necessary for the generation of antibodies. Animals can be immunized with cells bearing the antigen of interest. Splenocytes can then be isolated from the immunized animals, and the splenocytes can be fused with a myeloma cell line to produce a hybridoma (e.g., Meyaard, L., et. al., LAIR-1 , a novel inhibitory receptor expressed on human mononuclear leukocytes, Immunity 1997, 7:283-290; Wright et al., Inhibition of chicken adipocyte differentiation by in vitro exposure to monoclonal antibodies against embryonic chicken adipocyte plasma membranes, Immunity 2000, 13:233-242; Preston, et al., The leukocyte/neuron cell surface antigen OX2 binds to a ligand on macrophages,) Eur. J. Immunol. 1997, 27:1911 -1918, Kaithamana et al., Induction of experimental autoimmune Graves' disease in BALB/c mice, J. Immunol. 1999, 163:5157-5164).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapeutic, diagnostic, kit or other purposes, and include antibodies coupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g., colloidal gold (e.g., Le Doussal et al., Enhanced in vivo targeting of an asymmetric bivalent hapten to double-antigen-positive mouse B cells with monoclonal antibody conjugate cocktails, J. Immunol. 1991 , 146:169-175; Gibellini et al., Extracellular HIV-1 Tat protein induces the rapid Ser133 phosphorylation and activation of CREB transcription factor in both Jurkat lymphoblastoid T cells and primary... , J. Immunol. 1998160:3891 -3898; Hsing and Bishop, Requirement for nuclear factor-KB activation by a distinct subset of CD40-mediated effector functions in B lymphocytes, J. Immunol. 1999, 162:2804-2811 ; Everts etal., Selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate, J. Immunol. 2002, 168:883-889).

Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (e.g., Owens, et al., Flow Cytometry Principles for Clinical Laboratory Practice, 1994; Givan Flow Cytometry, 2nd ed.; 2001 ; Shapiro, Practical Flow Cytometry, 2003). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available (Molecular Probes, Catalogue, 2003; Sigma-Aldrich, Catalogue, 2003.

Standard methods of histology of the immune system are described (e.g., Muller- Harmelink (ed.), Human Thymus: Histopathology and Pathology, 1986; Hiatt, et al., Color Atlas of Histology, 2000; Louis, et al., Basic Histology: Text and Atlas, 2002.

Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available (e.g., GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al., A comparison of signal sequence prediction methods using a test set of signal peptides, Bioinformatics 2000, 16: 741-742; Menne, K.M.L., et. al. A comparison of signal sequence prediction methods using a test set of signal peptides, Bioinformatics 2000, 16, 741-742; Wren, etal., SIGNAL-sequence information and GeNomic Analysis Comput. Methods Programs Biomed. 2002, 68:177- 181 ; von Heijne, Patterns of amino acids near signal-sequence cleavage sites, Ear. J. Biochem. 1983, 133:17-21 ; von Heijne, A new method for predicting signal sequence cleavage sites, Nucleic Acids Res. 1986, 14:4683-4690).

Therapeutic Methods and Uses

The disclosure provides therapeutic methods and uses comprising administering to the subject the combinations as described herein, optionally in further combination with other therapeutic or palliative agents.

In one aspect, the disclosure provides a method for treating a cancer in a subject comprising administering to the subject a combination therapy of the disclosure. In one aspect, the disclosure provides a method for treating a cancer comprising administering to a subject in need thereof an amount of a HPK1 inhibitor and an amount of a PD-1 axis binding antagonist, wherein the amounts together are effective in treating cancer, and wherein the HPK1 inhibitor is a compound of Formula I, II, or Ila, or a pharmaceutically acceptable salt thereof, disclosed herein. In some such embodiments the subject is a human. In some embodiments, the method involves the use of a HPK1 inhibitor described herein in combination with an anti-PD-L1 antibody.

In some embodiments, the method involves the use of 4-[(1R)-1-aminopropyl]-2- {6-[(5S)-5-methyl-6,7-dihydro-5H-pyrrolo[2, 1 -c][1 , 2, 4]triazol-3-y l]py rid in-2-y l}-6-[(2R)-2- methylpyrrolidin-1 -yl]-2,3-dihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof, in combination with an anti-PD-1 antibody selected from sasanlimab, nivolumab, pembrolizumab, pidilizumab, cemiplimab, tislelizumab, spartalizumab, sintilimab, MEDI-0680, BGB-108, or AGEN2034 genolimzumab, CBT- 502, camrelizumab, or a combination thereof.

Preferably, the method involves the use of 4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5- methyl-6,7-dihydro-5/-/-pyrrolo[2, 1 -c][1 , 2 ,4]triazol-3-y l]py rid in-2-y l}-6-[(2R)-2- methylpyrrolidin-1 -yl]-2,3-dihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof, in combination with sasanlimab.

Preferably, the method involves the use of 4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5- methyl-6,7-dihydro-5/-/-pyrrolo[2, 1 -c][1 , 2 ,4]triazol-3-y l]py rid in-2-y l}-6-[(2R)-2- methylpyrrolidin-1 -yl]-2,3-dihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof, in combination with nivolumab.

Preferably, the method involves the use of 4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5- methyl-6,7-dihydro-5H-pyrrolo[2, 1 -c][1 , 2 ,4]triazol-3-y l]py rid in-2-y l}-6-[(2 R)-2- methylpyrrolidin-1 -yl]-2,3-dihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof, in combination with pembrolizumab.

In some embodiments, the treatment results in sustained response in the individual after cessation of the treatment. The methods of this disclosure may find use in treating conditions where enhanced immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer. As such, a variety of cancers may be treated, or their progression may be delayed.

In some embodiments, the individual has cancer that is resistant (has been demonstrated to be resistant) to one or more PD-1 axis binding antagonists. In some embodiments, resistance to PD-1 axis binding antagonist includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment. In some embodiments, resistance to PD-1 axis binding antagonist includes progression of the cancer during treatment with the PD-1 axis binding antagonist. In some embodiments, resistance to PD-1 axis binding antagonist includes cancer that does not response to treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, the cancer is at early stage or at late stage.

In certain embodiments, the subject has received one, two, three, four, five or more prior cancer treatments. In other embodiments, the subject is treatment-naive. In some embodiments, the subject has progressed on other cancer treatments. In certain embodiments, the prior cancer treatment comprised an immunotherapy. In other embodiments, the prior cancer treatment comprised a chemotherapy. In some embodiments, the tumor has reoccurred. In some embodiments, the tumor is advanced. In some embodiments, the tumor is metastatic. In other embodiments, the tumor is not metastatic.

In some embodiments, the subject has received a prior therapy to treat the tumor and the tumor is relapsed or refractory. In some embodiments, the subject has received a prior immuno-oncology therapy to treat the tumor and the tumor is relapsed or refractory. In some embodiments, the subject has received more than one prior therapy to treat the tumor and the subject is relapsed or refractory.

In some embodiments of the methods as described herein, the cancer is a solid tumor.

In a further embodiment, the disclosure is related to a method for treating cancer, wherein the cancer is selected from the group consisting of the cancer is brain cancer, head/neck cancer (including squamous cell carcinoma of the head and neck (SCCHN)), prostate cancer, bladder cancer (including urothelial carcinoma, also known as transitional cell carcinoma (TCC)), lung cancer (including squamous cell carcinoma, small cell lung cancer (SCLC), and non-small cell lung cancer (NSCLC)), breast cancer, ovarian cancer, bone cancer, colorectal cancer, kidney cancer, liver cancer (including hepatocellular carcinoma (HCC)), pancreatic cancer, esophageal cancer (including squamous cell carcinoma (SCC)), gastric cancer, gastroesophageal junction cancer, thyroid cancer, cervical cancer, uterine cancer, and/or renal cancer. In a further embodiment, the disclosure is related to a method for treating cancer, wherein the cancer is selected from the group consisting of squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), urothelial cancer and gastric cancer. In some embodiments, the disclosure is related to a method for treating cancer, wherein the cancer is melanoma.

In another embodiments, the disclosure provides a method for treating melanoma or breast cancer in a subject comprising administering to the subject 4-[(1 R)-1 - aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2, 1 -c][ 1 ,2,4]triazol-3-yl]pyridin- 2-yl}-6-[(2R)-2-methylpyrrolidin-1 -y l]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof, in combination with sasanlimab, nivolumab, or pembrolizumab.

In another embodiments, the disclosure provides a method for treating melanoma or breast cancer in a subject comprising administering to the subject 4-[(1 R)-1 - aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2, 1 -c][ 1 ,2,4]triazol-3-yl]pyridin- 2-yl}-6-[(2R)-2-methylpyrrolidin-1 -yl]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof, in combination with sasanlimab or nivolumab.

In one embodiment, the disclosure provides a method for treating melanoma in a subject comprising administering to the subject 4-[(1R)-1 -aminopropyl]-2-{6-[(5S)-5- methyl-6,7-dihydro-5/-/-pyrrolo[2, 1 -c][ 1 , 2 ,4]triazol-3-y l]py rid in-2-y l}-6-[(2R)-2- methylpyrrolidin-1 -yl]-2,3-dihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof, in combination with sasanlimab.

In another embodiments, the disclosure provides a method for treating melanoma or breast cancer in a subject comprising administering to the subject 4-[(1 R)-1 - aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2, 1 -c][ 1 ,2,4]triazol-3-yl]pyridin- 2-yl}-6-[(2R)-2-methylpyrrolidin-1 -yl]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof, in combination with nivolumab.

In another embodiments, the disclosure provides a method for treating melanoma or breast cancer in a subject comprising administering to the subject 4-[(1 R)-1 - aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2, 1 -c][ 1 ,2,4]triazol-3-yl]pyridin- 2-yl}-6-[(2R)-2-methylpyrrolidin-1 -yl]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof, in combination with pembrolizumab.

In some embodiments, the methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, phototherapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is immunotherapy.

In some embodiments, the methods of the present disclosure further comprise administering chemotherapy, radiotherapy, immunotherapy, or phototherapy, or any combinations thereof to the subject.

In some embodiments of the methods, uses, compositions, and kits described above and herein, the treatment further comprises administering a chemotherapeutic agent for treating or delaying progression of cancer in a subject. In some embodiments, the subject has been treated with a chemotherapeutic agent before the combination treatment with the HPK1 inhibitor, and PD-1 axis binding antagonist. In some embodiments, the subject treated with the combination of the HPK1 inhibitor, PD-1 axis binding antagonist, is refractory to a chemotherapeutic agent treatment. Some embodiments of the methods, uses, compositions, and kits described throughout the application, further comprise administering a chemotherapeutic agent for treating or delaying progression of cancer.

In some embodiments, the combination therapy of the disclosure comprises administration of a HPK1 inhibitor in combination with a PD-1 axis binding antagonist. In the methods provided herein, each of the HPK1 inhibitor, and PD-1 axis binding antagonist, may be administered in any suitable manner known in the art. In one embodiment, the HPK1 inhibitor and the PD-1 axis binding antagonist are administered simultaneously or sequentially in either order.

In some embodiments of each of the foregoing, the PD-1 axis binding antagonist is: a PD-1 antagonist; a PD-L1 antagonist; or a PD-1 antagonist and a PD-L1 antagonist.

In some embodiments of the each of the foregoing, a. the PD-1 binding antagonist and the PD-L1 binding antagonist are in the same composition. In one aspect, the disclosure provides a combination which is synergistic. In some such embodiments, the disclosure provides a synergistic combination comprising: (i) a HPK1 inhibitor of Formula I, II or Ila, or a pharmaceutically acceptable salt thereof; and (ii) a PD-1 axis binding antagonist described herein; for use in the treatment of cancer in a subject, wherein component (i) and component (ii) are synergistic. In some embodiments, the PD-1 axis binding antagonist is a PD-1 antagonist. In some embodiments, the PD-1 axis binding antagonist is a PD-L1 antagonist.

In some embodiments, the disclosure provides a synergistic combination comprising: (i) a HPK1 inhibitor of Formula I, II or Ila, or a pharmaceutically acceptable salt thereof; and (ii) sasanlimab; for use in the treatment of cancer in a subject, wherein component (i) and component (ii) are synergistic.

In a specific embodiment, the disclosure provides a combination comprising: (i) 4- [(1R)-1-aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-py rrolo[2,1 -c][1 ,2,4]triazol-3- y l]py rid in-2-y l}-6-[(2R)-2-m ethy Ipyrrol id in-1 -y l]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 - one, or a pharmaceutically acceptable salt thereof; and (ii) an anti-PD-1 antibody; for use in the treatment of cancer in a subject.

In a specific embodiment, the disclosure provides a combination comprising: a. (i) 4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2 ,1 -c][1 ,2,4]triazol-3- y l]py rid in-2-y l}-6-[(2R)-2-m ethy Ipyrrol id in-1 -yl]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 - one, or a pharmaceutically acceptable salt thereof; and (ii) sasanlimab; for use in the treatment of cancer in a subject.

In another embodiment, the disclosure provides a combination comprising: a. (i) 4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2 ,1 -c][1 ,2,4]triazol-3- y l]py rid in-2-y l}-6-[(2R)-2-m ethy Ipyrrol id in-1 -yl]-2 , 3-d ihydro-1 H-pyrrolo[3,4-c]pyridin-1 - one, or a pharmaceutically acceptable salt thereof; and (ii) nivolumab; for use in the treatment of cancer in a subject.

In another specific embodiment, the disclosure provides a combination comprising: a. (i) 4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2 ,1- c][1 ,2 ,4]triazol-3-y l]pyrid in-2-yl}-6-[(2R)-2-m ethy Ipyrrol idin-1 -yl]-2 , 3-d ihydro-1 H- pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof; and (ii) pembrolizumab; for use in the treatment of cancer in a subject. In a particular embodiment of each of the foregoing, the disclosure provides a combination wherein the PD-1 antagonist is an anti-PD-1 antibody; and/or the PD-L1 antagonist is an anti-PD-L1 antibody.

In some embodiments of the methods, uses, compositions, and kits described herein, the cancer is resistant to a therapeutic agent or class (including a standard of care agent or class). In some embodiments of the methods, uses, compositions, and kits described herein, the cancer is characterized by innate or acquired resistance to a therapeutic agent or class (including a standard of care agent or class).

In some embodiments of the methods, uses, compositions, and kits described herein, the cancer is refractory, i.e. , the cancer does not respond at all to treatment with a therapeutic agent or class (including a standard of care agent or class) or initially responds but starts to grow again in a very short period of time.

In some embodiments, the methods, uses, and combinations described herein relate to therapy, adjuvant therapy, first-line therapy, second-line therapy, or third-line or later lines of therapy. In some embodiments, the methods, uses, and combinations described herein relate to therapy. In some embodiments, the methods, uses, and combinations described herein relate to adjuvant therapy. In some embodiments, the methods, uses, and combinations described herein relate to first-line therapy. In some embodiments, the methods, uses, and combinations described herein relate to second- line therapy. In some embodiments, the methods, uses, and combinations described herein relate to third-line or later lines of therapy.

V. Dosage Forms and Regimens

Administration of the compounds of the disclosure may be affected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration.

Those skilled in the art will be able to determine the appropriate amount, dose or dosage of each compound, as used in the combination of the present disclosure, to administer to a patient, taking into account variety of factors, including, though not limited to, the degree of advancement of the disease, age, weight, general health, gender, diet, the compound administered, the time and route of administration, the nature and advancement of cancer, requiring treatment, and other medications the individual is taking.

In some embodiments, the methods of administration of the agents and combinations herein may include oral, intravenous, intramuscular subcutaneous, topical, transdermal, intraperitoneal, by implantation, by inhalation, intrathecal, intraventricular, or intranasal administration.

Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well- known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present disclosure.

For combination therapies as described herein, the agents may be administered at their approved dosages. Treatment is continued as long as clinical benefit is observed or until unacceptable toxicity or disease progression occurs. Nevertheless, in certain embodiments, the combination therapies of the present disclosure may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies. For example, the dosages of the agents administered are significantly lower than the approved dosage, e.g., a subtherapeutic dosage of the HPK1 inhibitor is administered in combination with a subtherapeutic dosage of a PD-1 axis binding antagonist. It will be appreciated by the skilled practitioner that when the agents of the disclosure are used as part of a combination therapy, a lower dosage of the agent may be desirable than when the agent alone is administered to a subject, a synergistic therapeutic effect may be achieved through the use of combination therapy which, in turn, permits use of a lower dose of the agent to achieve the desired therapeutic effect.

In one embodiment, the dosages may be lower and may also be applied less frequently, which may diminish the incidence or seventy of side-effects. This is in accordance with the desires and requirements of the subjects to be treated.

In one embodiment, the disclosure provides a pharmaceutical composition comprising an amount, which may be jointly therapeutically effective at treating cancer. In this pharmaceutical composition, two or more compounds may be administered together, one after the other or separately in one combined unit dosage form or in two separate unit dosage forms.

The unit dosage form may also be a fixed combination. It is to be noted that dosage values may vary with the type and seventy of the condition to be alleviated and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present disclosure encompasses intra-patient doseescalation as determined by the skilled artisan. Determining appropriate dosages and regimens for administration of the chemotherapeutic agent are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.

The amount of the agent of the disclosure administered will be dependent on the subject being treated, the seventy of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician.

An effective amount of the HPK1 inhibitor, and PD-1 axis binding antagonist, may be administered for prevention or treatment of disease. The appropriate dosage of the HPK1 inhibitor, and PD-1 axis binding antagonist, may be determined based on the type of disease to be treated, the HPK1 inhibitor, the PD-1 axis binding antagonist, the severity and course of the disease, the clinical condition of the subject, the subject's clinical history and response to the treatment, and the discretion of the attending physician. In some embodiments, combination treatment with HPK1 inhibitor, and PD-1 axis binding antagonist (e.g., anti- PD-1 antibody or anti-PD-L1 antibody), are synergistic, whereby an efficacious dose of the HPK1 inhibitor, PD-1 axis binding antagonist, in the combination is reduced relative to efficacious dose of the each of the HPK1 inhibitor, PD-1 axis binding antagonist, as a single agent.

In some embodiments, the subject has an advanced or metastatic solid tumor which has progressed following systemic anticancer therapy, including at least one checkpoint inhibitor (e.g., PD-1 , PD-L1 , or CTLA-4). In some embodiments, the subject has an advanced or metastatic solid tumor which displays resistance to one or more checkpoint inhibitors (e.g., PD-1 , PD-L1 , or CTLA-4). In some embodiments, the subject has an advanced or metastatic solid tumor which has progressed following systemic anticancer therapy, including at least one checkpoint inhibitor (e.g., PD-1 , PD-L1 , or CTLA-4), and displayed resistance to checkpoint inhibitors. In some such embodiments, the advanced or metastatic tumor status has been histologically or cytologically confirmed.

In some embodiments of each of the foregoing, the solid tumor is selected from the group consisting of gastric/gastroesophageal junction (GEJ) cancer, head and neck squamous cell carcinoma (HNSCC), and urothelial cancer. In some embodiments of each of the foregoing, the solid tumor is selected from the group consisting of non-small cell lung cancer and other solid tumors. In some embodiments, the solid tumor is advanced solid tumor. In some other embodiments, the solid tumor is metastatic solid tumor. In some embodiments, the PD-1 axis binding antagonist is sasanlimab and may be administered subcutaneously at a dose of about 1 , 2, 3, 4, 5, 6, 7 or 8 mg/kg at intervals of about 14 days (± 2 days) or about 21 days (± 2 days) or about 30 days (± 2 days) throughout the course of treatment. In some embodiment, sasanlimab is administer as a flat dose of about 80, 150, 160, 200, 240, 250, 300, 320, 350, or 400 mg.

VI. Kits

In some embodiments, the disclosure provides a kit comprising: (i) a pharmaceutical composition comprising a HPK1 inhibitor described herein and a pharmaceutically acceptable carrier; (ii) a pharmaceutical composition comprising a PD- 1 axis binding antagonist described herein and a pharmaceutically acceptable carrier.

In some embodiments, the kit further comprises package insert comprising instructions for using the HPK1 inhibitor in conjunction with PD-1 axis binding antagonist (e.g., anti-PD-1 or anti-PD-L1 antibody), treat or delay progression of cancer in a subject or to enhance immune function of a subject having cancer. In further embodiment, any of the HPK1 inhibitors, PD-1 axis binding antagonists, described herein may be included in the kits. For example, in some embodiments, the HPK1 inhibitor is a compound of Formula I, II, or Ila, or a pharmaceutically acceptable salt thereof. In some such embodiments, the HPK1 inhibitor is 4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5-methyl-6,7- dihydro-5/-/-pyrrolo[2, 1 -c][1 , 2 , 4]triazol-3-y l]py ridin-2-y l}-6-[(2R)-2-m ethy Ipy rrol id in-1 -y I]- 2,3-dihydro-1 H-pyrrolo[3,4-c]pyridin-1 -one, or a pharmaceutically acceptable salt thereof. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is selected from sasanlimab, nivolumab, pembrolizumab, pidilizumab, cemiplimab, tislelizumab, spartalizumab, sintilimab, MEDI- 0680, BGB-108, or AGEN2034 genolimzumab, CBT-502, camrelizumab, or a combination thereof. In one embodiment, the PD-1 binding antagonist is sasanlimab. In one embodiment, the PD-1 binding antagonist is nivolumab.

In some embodiments, the HPK1 inhibitor and the PD-1 axis binding antagonist are stored in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the kit further includes one or more of another agent (e.g. , a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.

The specification is sufficient to enable one skilled in the art to practice the disclosure. Various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES

The disclosure will be better understood with reference to the following examples. These examples are intended to representative of specific embodiments of the disclosure and are not intended as limiting the scope of the disclosure.

Preparation of Synthetic Intermediates

For the intermediates and Examples prepared herein, where the stereochemistry is known, the stereochemistry is as drawn and the name designates specific stereochemistry as (R) or (S). The stereochemistry as assigned herein is known because the compound was synthesized from known, chiral starting materials, or racemic mixtures were separated and the stereochemistry of certain examples or intermediates was confirmed using X-ray crystallography.

Intermediate 2: (5S)-3-(6-bromopyridin-2-yl)-5-methyl-6,7-dihydro-5/-/- pyrrolo[2, 1 -c][1 ,2,4]triazole Step 1 : te/t-buty I {(2S)-5-[2-(6-bromopyridine-2-carbonyl)hydrazinyl]-5- oxopentan-2-yl}carbamate (2a)

Boc 2a

A solution of (4S)-4-[(fe/t-butoxycarbonyl)amino]pentanoic acid (600 mg, 2.76 mmol) in THF (13.8 mL, 0.2 M) was cooled to 0 °C. Propylphosphonic anhydride solution (50% solution in EtOAc, 3.62 mL, 6.08 mmol) was added to the solution at 0 °C before the bath was removed and the reaction mixture was stirred for 30 min at RT. Then, A/,/V- diisopropylethylamine (2.89 mL, 16.6 mmol) and 6-bromopicolinohydrazide (656 mg, 3.04 mmol) were added and the reaction mixture was stirred at RT for 22 h. LCMS analysis showed consumption of the starting material. The reaction was quenched with water (15 mL) and transferred to a separatory funnel with EtOAc (20 mL). The layers were separated, and the organic phase was washed sequentially with 20% citric acid (20 mL), a saturated solution of NaHCOs (20 mL), and brine (20 mL). The organic extract was then dried over MgSO4, filtered, and concentrated to dryness to provide the title compound (2a) (1.07 g, 93% yield) as an off-white solid, which was taken on without further purification. ¹ H NMR (400 MHz, CDCh) 5 9.76 (br. s, 1 H), 9.36 (br. s, 1 H), 8.15 (dd, J = 0.9, 7.5 Hz, 1 H), 7.77 - 7.71 (m, 1 H), 7.69 - 7.64 (m, 1 H), 4.45 (br. d, J = 1.0 Hz, 1 H), 3.91 (br. s, 1 H), 2.47 - 2.35 (m, 2H), 2.00 - 1 .89 (m, 1 H), 1 .75 - 1 .66 (m, 1 H), 1 .48 (s, 9H), 1 .22 (d, J = 6.6 Hz, 3H). LCMS m/z (APCI) for (CnHisBrN^), 315.0 (M+H- Boc) ⁺ .

Step 2: te/t-butyl {(2S)-4-[5-(6-bromopyridin-2-yl)-1 ,3,4-oxadiazol-2-yl]butan-2- yljcarbamate (2b)

To a solution of te/t-butyl {(2S)-5-[2-(6-bromopyridine-2-carbonyl)hydrazinyl]-5- oxopentan-2-yl}carbamate (2a) (290 mg, 0.698 mmol) in DCM (2.8 mL, 0.25 M) was added triethylamine (0.292 mL, 2.09 mmol) and p-toluenesulfonyl chloride (160 mg, 0.838 mmol). The reaction was stirred at RT for 16 h. LCMS analysis showed consumption of the starting material. Ethylenediamine (0.047 mL, 0.698 mmol) was added to scavenge excess p-toluenesulfonyl chloride; during the addition, a precipitate formed immediately. After stirring at RT for 30 min, the reaction was washed with 20% citric acid (5 mL) and the layers were separated. The aqueous layer was extracted with DCM (5 mL), then the combined organic layers were washed with brine (10 mL), dried over MgSO4, filtered and concentrated to dryness to provide the title compound (2b) (274 mg, 98% yield) as a light yellow solid, which was taken on without further purification. ¹ H NMR (400 MHz, CDCh) 5 8.21 (dd, J = 0.9, 7.6 Hz, 1 H), 7.76 - 7.69 (m, 1 H), 7.68 - 7.65 (m, 1 H), 4.39 (br. s, 1 H), 3.85 (br. s, 1 H), 3.08 - 3.01 (m, 2H), 2.17 - 2.03 (m, 1 H), 2.03 - 1.90 (m, 1 H), 1.44 (s, 9H), 1 .22 (d, J = 6.6 Hz, 3H). LCMS m/z (APCI) for (CuH BrlS O), 297.0 (M+H-Boc) ⁺ .

Step 3: Intermediate 2

A microwave vial was charged with te/Y-butyl {(2S)-4-[5-(6-bromopyridin-2-yl)- 1 ,3,4-oxadiazol-2-yl]butan-2-yl}carbamate (2b) (150 mg, 0.378 mmol) and trifluoroethanol (1 .89 mL, 0.2 M) and was sealed before heating in the microwave to 180 °C for 30 min. LCMS analysis showed consumption of the starting material. The reaction mixture was concentrated, and the residue was purified by flash chromatography (SiC>2, 100% heptane to 1 :10 MeOH/EtOAc) to provide Intermediate 2 (74.3 mg, 71 % yield) as a tan, gummy solid. ¹ H NMR (400 MHz, CDCh) 5 8.38 (d, J = 7.7 Hz, 1 H), 7.74 (t, J = 7.8 Hz, 1 H), 7.58 (d, J = 7.9 Hz, 1 H), 5.22 - 5.08 (m, 1 H), 3.21 - 3.16 (m, 1 H), 3.16 - 3.02 (m, 2H), 2.55 - 2.44 (m, 1 H), 1 .60 (d, J = 6.6 Hz, 3H). LCMS m/z (APCI) for (CnHiiBrN ₄ ), 279.1 (M+H) ⁺ . Determined to be a single enantiomer by SFC (10-60% methanol (0.5% NH3) in carbon dioxide @ 400-450 bar, gradient time = 2 min, flow rate = 4 mL/min, Chiralpack IC-U 50mm*3mm*1 ,6pm column). Stereochemistry of Intermediate 2 was assigned based on use of (4S)-4-[(fe/Y-butoxycarbonyl)amino]pentanoic acid in step 1 .

Intermediate 3: (5S)-3-(6-bromopyridin-2-yl)-5-ethyl-6,7-dihydro-5/-/-pyrrol o[2, 1 - c][1 ,2,4]triazole Step 1 : te/Y-butyl {(2S)-1 -[methoxy(methyl)amino]-1 -oxobutan-2-yl}carbamate (3a)

A solution of (2S)-2-[(tert-butoxycarbonyl)amino]butanoic acid (2.0 g, 9.84 mmol) in THF (49.2 mL, 0.2M) was cooled to 0 °C. Propylphosphonic anhydride solution (50% solution in EtOAc, 12.9 mL, 21 .6 mmol) was added to the solution at 0 °C before the bath was removed and the reaction mixture was allowed to warm to RT and stirred for 30 min. Then, /V,/V-di isopropylethylamine (10.3 mL, 59.0 mmol) and methoxy(methyl)amine hydrochloride (1 .06 g, 10.8 mmol) were added and the reaction mixture was stirred at RT for 18 h. LCMS analysis showed consumption of the starting material. The reaction was quenched with water (40 mL) and transferred to a separatory funnel with EtOAc (40 mL). The layers were separated, and the organic phase was washed sequentially with 20% citric acid (40 mL), a saturated solution of NaHCOs (40 mL), and brine (40 mL). The organic extract was then dried over MgSO4, filtered, and concentrated to dryness to provide the title compound (3a) (1 .29 g, 53% yield) as a yellow oil, which was taken on without further purification. ¹ H NMR (400 MHz, CDCh) 5 5.28 - 5.12 (m, 1 H), 4.74 - 4.52 (m, 1 H), 3.79 (s, 3H), 3.23 (s, 3H), 1 .85 - 1 .73 (m, 1 H), 1 .64 - 1 .54 (m, 1 H), 1 .46 (s, 9H), 0.96 (t, J = 7.5 Hz, 3H). LCMS m/z (APCI) for (C11H22N2O4), 247.1 (M+H) ⁺ .

Step 2: te/t-butyl (S)-(1 -oxobutan-2-yl)carbamate (3b)

A solution of te/t-butyl {(2S)-1 -[methoxy(methyl)amino]-1 -oxobutan-2- yljcarbamate (3a) (1 .29 g, 5.25 mmol) in THF (26.2 mL, 0.2M) was cooled to 0 °C. Lithium aluminum hydride (2.3 M solution in 2-MeTHF, 2.51 mL, 5.77 mmol) was added dropwise to the solution at 0 °C. The reaction mixture was stirred for 40 min at 0 °C. LCMS analysis showed consumption of the starting material. The reaction was quenched with EtOAc (10 mL) and 1 M HCI (10 mL) and transferred to a separatory funnel. The layers were separated, and the aqueous phase was extracted with EtOAc (3 x 10 mL). The combined organic extracts were then dried over MgSO4, filtered, and concentrated to dryness. The residue was purified by flash chromatography (SiO2, 100% heptane to 100% EtOAc) to provide to provide the title compound (3b) (0.575 g, 59% yield) as a white solid. ¹ H NMR (400 MHz, CDCIs) 5 9.60 (s, 1 H), 5.09 (br. s, 1 H), 4.22 (br. s, 1 H), 2.04 - 1.90 (m, 1 H), 1 .73 - 1 .64 (m, 2H), 1 .48 (s, 9H), 0.99 (t, J = 7.5 Hz, 3H).

Step 3: ethyl (2E,4S)-4-[(tert-butoxycarbonyl)amino]hex-2-enoate (3c)

To a solution of te/Y-butyl (S)-(1 -oxobutan-2-yl)carbamate (3b) (575 mg, 3.07 mmol) in DCM (6.14 mL, 0.5 M) was added (ethoxycarbonylmethylene)triphenylphosphorane (1.60 g, 4.61 mmol). The reaction mixture was stirred at RT for 17 h. LCMS analysis showed consumption of the starting material. The reaction was concentrated to dryness before isopropanol (8.5 mL, 0.36 M) and zinc chloride (1.26 g, 9.21 mmol) were added to precipitate the triphenylphosphine oxide. After stirring at RT for 30 min, the reaction mixture was filtered and concentrated to dryness. The residue was purified by flash chromatography (SiC>2, 100% heptane to 100% EtOAc) to provide the title compound (3c) (653 mg, 83% yield, >20:1 ratio of E/Z isomers) as a colorless oil. ¹ H NMR (400 MHz, CDCI3) 5 6.86 (dd, J = 5.4, 15.6 Hz, 1 H), 5.94 (dd, J = 1.7, 15.7 Hz, 1 H), 4.50 (br. s, 1 H), 4.31 - 4.16 (m, 3H), 1.65 - 1.63 (m, 1 H), 1 .60 - 1 .51 (m, 1 H), 1 .47 (s, 9H), 1 .32 - 1 .30 (m, 3H), 0.99 - 0.95 (m, 3H). LCMS m/z (APCI) for (C8H15NO2), 158.2 (M+H-Boc) ⁺ .

Step 4: ethyl (4S)-4-[(tert-butoxycarbonyl)amino]hexanoate (3d) EtC^C^^s^Et

NHBoc 3d

To a solution of ethyl (2E,4S)-4-[(tert-butoxycarbonyl)amino]hex-2-enoate (3c) (653 mg, 2.54 mmol) in methanol (12.7 mL, 0.2 M) was added palladium on carbon (10% w/w, 270 mg, 0.254 mmol). The reaction vial was evacuated and refilled with H2 under dynamic vacuum for 10 seconds. Then the reaction mixture was stirred at RT under 1 atm H2 for 18 h. The mixture was stirred at RT for 17 h. LCMS analysis showed consumption of the starting material. The reaction was filtered over Celite® and concentrated to dryness. The residue was purified by flash chromatography (SiC>2, 100% heptane to 100% EtOAc) to provide the title compound (3d) (589 mg, 90% yield) as a colorless oil. ¹ H NMR (400 MHz, CDCI3) 5 4.37 - 4.23 (m, 1 H), 4.20 - 4.11 (m, 2H), 3.61 - 3.46 (m, 1 H), 2.42 - 2.33 (m, 2H), 1 .93 - 1 .82 (m, 1 H), 1.71 - 1 .62 (m, 1 H), 1.57 - 1 .51 (m, 1 H), 1 .46 (s, 9H), 1 .44 - 1 .38 (m, 1 H), 1 .30 - 1 .26 (m, 3H), 0.94 (t, J = 7.4 Hz, 3H).

Step 5: (4S)-4-[(tert-butoxycarbonyl)amino]hexanoic acid (3e) HO ₂ C^^^Et

NHBoc 3e

To a solution of ethyl (4S)-4-[(tert-butoxycarbonyl)amino]hexanoate (3d) (589 mg, 2.27 mmol) in THF (11 .4 mL, 0.2 M) and MeOH (5.7 mL, 0.4 M) was added a solution of LiOH (544 mg, 22.7 mmol) in water (2.84 mL, 0.8 M). The reaction mixture was stirred at RT for 24 h. The reaction was concentrated before the addition of water and 1 M HCI until a pH of 5 was reached. The aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine, dried over MgSC , filtered, and concentrated to dryness to provide the title compound (3e) (260 mg, 50% yield) as a lightyellow oil, which was taken on without further purification.

Step 6: te/Y-butyl {(3S)-6-[2-(6-bromopyridine-2-carbonyl)hydrazinyl]-6-oxohexa n- 3-yl}carbamate (3f)

A solution of (4S)-4-[(tert-butoxycarbonyl)amino]hexanoic acid (3e) (260 mg, 1.12 mmol) in THF (5.6 mL, 0.2 M) was cooled to 0 °C. Propylphosphonic anhydride solution (50% solution in EtOAc, 1.47 mL, 2.47 mmol) was added to the solution at 0 °C before the bath was removed and the reaction mixture was stirred for 30 min at RT. Then, N,N- diisopropylethylamine (1.17 mL, 6.74 mmol) and 6-bromopicolinohydrazide (267 mg, 1 .24 mmol) were added and the reaction mixture was stirred at RT for 18 h. The reaction was quenched with water (15 mL) and transferred to a separatory funnel with EtOAc (20 mL). The layers were separated, and the organic phase was washed sequentially with 20% citric acid (20 mL), a saturated solution of NaHCOs (20 mL), and brine (20 mL). The organic extract was then dried over MgSO4, filtered, and concentrated to dryness. The residue was purified by flash chromatography (SiO2, 100% heptane to 100% EtOAc) to provide the title compound (3f) (241 mg, 50% yield) as a white solid. ¹ H NMR (400 MHz, CDCIs) 5 9.76 (br. s, 1 H), 9.29 (br. s, 1 H), 8.15 (d, J = 6.8 Hz, 1 H), 7.76 - 7.71 (m, 1 H), 7.68 - 7.64 (m, 1 H), 4.45 - 4.31 (m, 1 H), 3.69 (br. s, 1 H), 2.45 - 2.37 (m, 2H), 2.04 - 1 .94 (m, 1 H), 1 .69 - 1 .62 (m, 1 H), 1 .49 (s, 9H), 1 .45 (br. d, J = 7.3 Hz, 2H), 0.99 (t, J = 7.5 Hz, 3H). LCMS m/z (APCI) for (Ci2Hi7BrN ₄ O ₂ ), 329.0 (M+H-Boc) ⁺ .

Step 7: te/t-butyl {(3S)-1-[5-(6-bromopyridin-2-yl)-1 ,3,4-oxadiazol-2-yl]pentan-3- yljcarbamate (3g)

To a solution of te/t-butyl {(3S)-6-[2-(6-bromopyridine-2-carbonyl)hydrazinyl]-6- oxohexan-3-yl}carbamate (3f) (241 mg, 0.561 mmol) in DCM (2.2 mL, 0.25 M) was added triethylamine (0.235mL, 1.68 mmol) and p-toluenesulfonyl chloride (128 mg, 0.673 mmol). The reaction was stirred at RT for 16 h. LCMS analysis showed consumption of the starting material. Ethylenediamine (0.038 mL, 0.561 mmol) was added to scavenge excess p-toluenesulfonyl chloride; during the addition a precipitate formed immediately. After stirring at RT for 30 min, the reaction was washed with 20% citric acid (5 mL) and the layers were separated. The aqueous layer was extracted with DCM (5 mL) then the combined organic layers were washed with brine (10 mL), dried over MgSO ₄ , filtered and concentrated to dryness. The residue was purified by flash chromatography (SiO ₂ , 100% heptane to 100% EtOAc) to provide the title compound (3g) (178 mg, 77% yield) as a white solid. ¹ H NMR (400 MHz, CDCh) 5 8.22 (dd, J = 0.7, 7.6 Hz, 1 H), 7.77 - 7.72 (m, 1 H), 7.68 - 7.65 (m, 1 H), 4.35 (br. d, J = 8.2 Hz, 1 H), 3.65 (br. s, 1 H), 3.07 (dt, J = 6.1 , 10.0 Hz, 2H), 2.21 - 2.11 (m, 1 H), 1.93 - 1.83 (m, 1 H), 1.65 - 1.61 (m, 1 H), 1.53 - 1.48 (m, 1 H), 1 .46 (s, 9H), 0.98 (t, J = 7.4 Hz, 3H). LCMS m/z (APCI) for (Ci ₂ Hi ₅ BrN ₄ O), 311 .0 (M+H-Boc) ⁺ .

Step 8: Intermediate 3

A microwave vial was charged with te/Y-butyl {(3S)-1 -[5-(6-bromopyridin-2-yl)- 1 ,3,4-oxadiazol-2-yl]pentan-3-yl}carbamate (3g) (178 mg, 0.431 mmol) and trifluoroethanol (2.16 mL, 0.2 M) and was sealed before heating in the microwave to 180 °C for 60 min. LCMS analysis showed consumption of the starting material. The reaction mixture was concentrated, and the residue was purified by flash chromatography (SiC>2, 100% heptane to 1 :10 MeOH/EtOAc) to provide Intermediate 3 (116 mg, 91 % yield) as a colorless oil. ¹ H NMR (400 MHz, CDCh) 5 8.26 (dd, J = 0.7, 7.7 Hz, 1 H), 7.67 (t, J = 7.9 Hz, 1 H), 7.49 (dd, J = 0.7, 7.8 Hz, 1 H), 4.90 - 4.83 (m, 1 H), 3.07 - 2.89 (m, 3H), 2.58 - 2.49 (m, 1 H), 2.04 - 1 .97 (m, 1 H), 1 .83 - 1 .70 (m, 1 H), 0.96 (t, J = 7.5 Hz, 3H). LCMS m/z (APCI) for (Ci2Hi ₃ BrN ₄ ), 293.0 (M+H) ⁺ . Determined to be 97.4% ee by SFC (10-60% methanol (0.5% NH3) in carbon dioxide @ 400-450 bar, gradient time = 2 min, flow rate = 4 mL/min, Kromasil (R,R)Whelk-0 50mm*3mm*1 ,8pm column). Stereochemistry was assigned based on use of (2S)-2-[(tert-butoxycarbonyl)amino]butanoic acid in the first step.

Intermediate 4: (S, S)-2-methyl-N-[(1 R)-1 -{6-[(2R)-2-methylpyrrolidin-1 -y l]-1 -oxo- 2,3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}ethyl]propane-2-sulfinamide

Step 1 : {2-chloro-6-[(2R)-2-methylpyrrolidin-1 -yl]pyridin-4-yl}(piperidin-1 - yl)methanone (4a)

A solution of (2,6-dichloropyridin-4-yl)(piperidin-1 -yl)methanone (600 mg, 2.32 mmol) and (2R)-2-methylpyrrolidine (591 mg, 6.95 mmol) in DMF (1.5 mL) was stirred at 100 °C for 16 h. LCMS analysis showed consumption of the starting material. The reaction was cooled to RT, H2O (40 mL) was added, and the reaction was extracted with DCM (3 x4 OmL). The combined organic layers were dried over Na2SO ₄ , filtered, and concentrated. The residue was purified by flash chromatography (24 g SiC>2, 0-20% EtOAc/heptane) to provide the title compound (4a) (664 mg, 93% yield). ¹ H NMR (400 MHz, CDCIs) 5 6.44 (d, J = 1.0 Hz, 1 H), 6.21 (d, J = 1.0 Hz, 1 H), 4.12 (q, J = 7.1 Hz, 1 H),

3.72 - 3.62 (m, 2H), 3.54 (ddd, J = 10.5, 7.6, 2.9 Hz, 1 H), 3.40 - 3.28 (m, 2H), 2.10 - 2.04 (m, 2H), 1 .75 - 1 .62 (m, 4H), 1 .26 (t, J = 7.2 Hz, 1 H), 1 .21 (d, J = 6.3 Hz, 2H); m/z (APCI+) for (C16H22CIN3O), 308.2 (M+H) ⁺ .

Step 2: 2-chloro-6-[(2R)-2-methylpyrrolidin-1 -y l]~4-(piperid ine-1 -carbonyl)pyridine- 3-carbaldehyde (4b)

To a solution of DMF (473 mg, 6.47 mmol) in DCM (3.0 mL) was added POCI3 (992 mg, 6.47 mmol). The mixture was stirred for 10 min and then a solution of {2-chloro- 6-[(2R)-2-methylpyrrolidin-1 -yl]pyridin-4-yl}(piperidin-1-yl)methanone (4a) (664 mg, 2.16 mmol) in DCM (3.0 mL) was added. The mixture was stirred at reflux for 15 h. LCMS analysis showed consumption of the starting material. The reaction was concentrated to dryness and slowly poured into a saturated solution of NaHCOs (30 mL). The mixture was extracted with DCM (3 x 30 mL). The combined organics were dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (24 g Si O2, 0-40% EtOAc/heptane) to provide the title compound (4b) (568 mg, 78 % yield). ¹ H NMR (400 MHz, CDCI ₃ ) 5 10.07 (S, 1 H), 5.99 (s, 1 H), 4.13 - 4.65 (m, 1 H), 3.68 - 3.83 (m, 1 H), 3.55 - 3.68 (m, 2H), 3.35 - 3.55 (m, 1 H), 2.98 - 3.20 (m, 2H), 1.88 - 2.17 (m, 3H), 1.71 - 1.83 (m, 2H), 1.55 - 1.67 (m, 3H), 1.46 - 1.55 (m, 1 H), 1.31 - 1.42 (m, 1 H), 1.17 - 1.26 (m, 3H); m/z (APCI+) for (C17H22CIN3O2), 336.1 (M+H) ⁺ .

Step 3: A/-[(E)-{2-chloro-6-[(2R)-2-methylpyrrolidin-1 -y l]-4-(piperid ine-1 - carbonyl)pyridin-3-yl}methylidene]-2-methylpropane-2-sulfina mide (4c)

A mixture of 2-chloro-6-[(2R)-2-methylpyrrolidin-1 -yl]-4-(piperidine-1 - carbonyl)pyridine-3-carbaldehyde (4b) (432 mg, 1.29 mmol), (R)-(+)-2-methyl-2- propanesulfinamide (187 mg, 1 .54 mmol), and Ti(0Et)4 (880 mg, 3.86 mmol) in THF (10.0 mL) was stirred at 45 °C for 16 h. LCMS analysis showed ~25% remaining starting material. Additional batches of (R)-(+)-2-methyl-2-propanesulfinamide (62.4 mg, 0.515 mmol), and Ti(0Et)4 (293 mg, 1 .29 mmol) were added and the mixture was stirred at 50 °C for 16 h. LCMS analysis showed consumption of the starting material. The reaction was cooled to RT. The mixture was diluted with DCM (50 mL) and washed with a saturated solution of NaHCOs (35 mL) and brine (35 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to provide the title compound (4c) (495 mg, 88% yield) as a white gum, which was taken on without further purification, m/z (APCI+) for (C21H31CIN4O2S), 440.2 (M+H) ⁺ .

Step 4: 4-chloro-6-[(2R)-2-methylpyrrolidin-1 -y l]-2 , 3-d ihydro-1 /-/-pyrrolo[3,4- c]pyridin-1-one (4d)

A solution of /V-[(E)-{2-chloro-6-[(2R)-2-methylpyrrolidin-1-yl]-4-(piperi dine-1 - carbonyl)pyridin-3-yl}methylidene]-2-methylpropane-2-sulfina mide (4c) (495 mg, 1.13 mmol) in THF (15.0 mL) was cooled to 0 °C and then a solution of LiBH4 (2.0 M in THF, 620 mL, 1 .24 mmol) was added. The mixture was stirred at 0 °C for 2 h and then a solution of NaOMe (25% in MeOH, 2.5 mL, 10.1 mmol) was added. The reaction was allowed warm to RT and then stirred for 16 h. The reaction was diluted with DCM (60 mL) and washed with saturated aqueous NH4CI (60 mL) and brine (60 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (SiC>2, 50-100% EtOAc/heptane) to provide the title compound (4d) (199 mg, 70% yield) as a colorless foam. ¹ H NMR (400 MHz, CDCI3) 5 6.68 (s, 1 H), 6.45 (s, 1 H), 4.35 (s, 2H), 4.21 - 4.14 (m, 1 H), 3.58 (ddd, J = 10.5, 7.6, 2.8 Hz, 1 H), 3.39 (q, J = 8.9 Hz, 1 H), 2.13 - 1.97 (m, 2H), 1.75 (dt, J = 5.2, 2.6 Hz, 1 H), 1.23 (d, J = 6.3 Hz, 3H). One hydrogen atom assumed obscured by water peak; m/z (APCI+) for (C12H14CIN3O), 252.3 (M+H) ⁺ .

Step 5: 6-[(2R)-2-methylpyrrolidin-1 -y l]-1 -oxo-2,3-dihydro-1 /-/-pyrrolo[3,4- c]pyridine-4-carbonitrile (4e)

A mixture of 4-chloro-6-[(2R)-2-methylpyrrolidin-1 -yl]-2,3-dihydro-1 /-/-pyrrolo[3,4- c]pyridin-1 -one (4d) (438 mg, 1.74 mmol), Zn(CN)2 (306 mg, 2.6 mmol), DMF (15 mL) and Pd(PPh3)4 (100 mg, 0.09 mmol) was heated to 140 °C in the microwave for 30 min. The reaction was diluted with DCM and filtered. The filter cake was washed with DCM. The filtrate was concentrated in vacuo and the crude compound was purified by flash chromatography (SiC>2, 0-100% 1 :1 EtOAc:DCM/heptane) to the title compound (4e) as a yellow solid (375 mg, 89% yield). ¹ H NMR (400 MHz, CDCh) 5 7.00 - 6.94 (m, 1 H), 6.46 - 6.32 (m, 1 H), 4.55 (s, 2H), 4.29 - 4.19 (m, 1 H), 3.61 (ddd, J = 2.6, 7.6, 10.3 Hz, 1 H), 3.46 - 3.34 (m, 1 H), 2.22 - 2.02 (m, 3H), 1.86 - 1 .72 (m, 1 H), 1 .25 (d, J = 6.4 Hz, 3H); m/z (APCI+) for (C13H14N4O), 243.1 (M+H) ⁺ .

Step 6: 4-acetyl-6-[(2R)-2-methylpyrrolidin-1 -y l]-2 , 3-d ihydro-1 /-/-pyrrolo[3,4- c]pyridin-1-one (4f)

To a solution of 6-[(2R)-2-methylpyrrolidin-1 -yl]-1 -oxo-2,3-dihydro-1 H-pyrrolo[3,4- c]pyridine-4-carbonitrile (4e) (355 mg, 1.47 mmol) in THF (15 mL) at ice/water bath was added MeMgBr (4.88 mL, 14.7 mmol, 3.0 M in THF). The resulting mixture was stirred at this temperature for 10 min, then allowed warmed to RT and allowed to stir for 2 h. The mixture was quenched with 2 N HCI (6 mL) at 0 °C and stirred at RT for 15 min. The mixture was neutralized with a saturated solution of NaHCOs and extracted with DCM (3 x 40 mL). The combined organic layers were dried over Na2SO4 filtered, and concentrated. The crude compound was purified by flash chromatography (S iC>2, 20-50% EtOAc/heptane) to provide the title compound (4f) as a yellow solid (190 mg, 50% yield). ¹ H NMR (400 MHz, CDCh) 6.98 (s, 1 H), 6.59 (br. s, 1 H), 4.70 (s, 2H), 4.28 (br. t, J = 5.7 Hz, 1 H), 3.64 (ddd, J = 2.6, 7.5, 10.1 Hz, 1 H), 3.48 - 3.38 (m, 1 H), 2.70 (s, 3H), 2.19 - 2.10 (m, 2H), 2.08 - 2.01 (m, 1 H), 1 .79 (td, J = 2.6, 5.0 Hz, 1 H), 1 .30 (d, J = 6.2 Hz, 3H); m/z (APCI+) for (C14H17N3O2), 260.2 (M+H) ⁺ .

Step 7: Intermediate 4

To a 40 mL vial was added 4-acetyl-6-[(2R)-2-methylpyrrolidin-1 -yl]-2,3-dihydro- 1 H-pyrrolo[3,4-c]pyridin-1 -one (4f) (200 mg, 0.77 mmol), (S)-tert-butylsulfinamide (187 mg, 1.54 mmol), THF (1.54 mL) and Ti(0Et)4 (1.54 mmol , 0.323 mL). The vial was capped and heated to 80 °C for 48 h. The reaction was then cooled to -78 °C (dry ice/acetone bath) and L-selectride (1.54 mmol, 1.54 mL, 1.0 M in THF) dropwise. The resulting mixture was stirred at -78 °C for 3 hr. The mixture was warmed to RT and quenched with saturated NH4CI (2 mL) dropwise, then added DCM (20 mL) and brine (20 mL). The mixture was filtered through Celite® and washed with DCM (40 mL). The organic layer was collected, and the aqueous layer was extracted with DCM (20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude was purified by flash chromatography (SiC>2, solvent 0-10% MeOH in EtOAc) to provide Intermediate 4 as a yellow foam (127 mg, 45% yield), m/z (APCI+) for (C18H28N4O2S), 365.3 (M+H) ⁺ . Stereochemistry was assigned based on use of (2R)-2- methylpyrrolidine in step 1 and (S)-tert-butylsulfinamide in step 7.

Intermediate 5: (S _; R)-2-methyl-A/-[(1 R)-1 -{6-[(2R)-2-methylpyrrolidin-1 -y l]-1 -oxo- 2,3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}propyl]propane-2-sulfinamide

Step 1 : 6-[(2R)-2-methylpyrrolidin-1 -yl]-4-propanoyl-2,3-dihydro-1 /-/-pyrrolo[3,4- c]pyridin-1-one (5a) A solution of 6-[(2R)-2-methylpyrrolidin-1 -yl]-1 -oxo-2,3-dihydro-1 /-/-pyrrolo[3,4- c]pyridine-4-carbonitrile (4e) (808 mg, 3.33 mmol) in THF (8.0 mL) was cooled to 0 °C and then treated with a solution of ethylmagnesium bromide (3.0 M in Et20, 11.1 mL, 33.3 mmol). The mixture was stirred at RT for 1 h. LCMS analysis showed consumption of the starting material. The reaction was cooled to 0 °C and quenched by addition of 2 N HCI. The mixture was stirred at RT for 10 min, neutralized by addition of a saturated solution of NaHCOs, and then extracted with DCM (2 x 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (24 g SiC>2, 0-100% EtOAc/heptane) to provide the title compound (5a) (646 mg, 71 % yield) as a yellow oil. ¹ H NMR (400 MHz, CDCh) 5 7.01 (s, 1 H), 6.41 (br. s, 1 H), 4.73 (s, 2H), 4.39 - 4.22 (m, 1 H), 3.66 (ddd, J = 2.6, 7.4, 10.1 Hz, 1 H), 3.52 - 3.38 (m, 1 H), 3.30 - 3.14 (m, 2H), 2.26 - 2.10 (m, 2H), 2.06 - 2.01 (m, 1 H), 1.81 (td, J = 2.5, 5.1 Hz, 1 H), 1.31 (d, J = 6.2 Hz, 3H), 1.24 (t, J = 7.3 Hz, 3H); m/z (APCI+) for (C15H19N3O2), 274.2 (M+H) ⁺ .

Step 2: (S,S)-2-methyl-A/-[(1 E)-1 -{6-[(2R)-2-methylpyrrolidin-1 -y l]-1 -oxo-2,3- dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}propylidene]propane-2-sulfina mide (5b)

To a solution of 6-[(2R)-2-methylpyrrolidin-1 -yl]-4-propanoyl-2,3-dihydro-1/-/- pyrrolo[3,4-c]pyridin-1 -one (5a) (640 mg, 2.34 mmol) in THF (2.0 mL) were added (S)- (-)-2-methyl-2-propanesulfinamide (568 mg, 4.68 mmol) and Ti(0Et)4 (2.14 g, 9.37 mmol). The mixture was stirred at 90 °C for 23 h. LCMS analysis showed consumption of the starting material. The mixture was cooled to RT and brine (40 mL) and DCM (30 mL) were then added. The mixture was stirred for 10 min and then filtered through Celite®. The layers were separated. The aqueous layer was extracted with DCM (30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated to provide the title compound (5b) (779 mg, 88% yield) as a white foam which was directly taken on in the next step, m/z (APCI+) for (C19H28N4O2S), 377.2 (M+H) ⁺ . Step 3: Intermediate 5

A solution of (S _; S)-2-methyl-A/-[(1 E)-1-{6-[(2R)-2-methylpyrrolidin-1-yl]-1 -oxo-2,3- dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}propylidene]propane-2-sulfina mide (5b) (654 mg, 1.40 mmol) in THF (12.0 mL) was cooled to -78 °C and then treated dropwise with a solution of L-selectride (1 .0 M in THF, 2.5 mL, 2.5 mmol). The mixture was stirred at -78 °C for 1.5 h. Additional L-selectride (1.0 M, 0.417 mL, 0.417 mmol) was added and the mixture was stirred at -78 °C for a further 1 .5 h. The mixture was quenched with MeOH, diluted with brine (50mL), and extracted with DCM (2 x 50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (12 g SiC>2, 0-10% MeOH/DCM) to provide Intermediate 5 (300 mg, 57% yield) as a yellow gum. ¹ H NMR (400 MHz, DMSO-de) 5 8.66 (s, 1 H), 6.48 (s, 1 H), 5.14 (d, J = 6.6 Hz, 1 H), 4.35 (s, 2H), 4.27 - 4.14 (m, 2H), 3.59 - 3.44 (m, 1 H), 3.29 - 3.18 (m, 1 H), 2.14 - 2.01 (m, 2H), 1.98 - 1.81 (m, 3H), 1.73 - 1.60 (m, 1 H), 1.20 (d, J = 6.2 Hz, 3H), 1.07 (s, 9H), 0.86 - 0.73 (m, 3H); m/z (APCI+) for (C19H30N4O2S), 379.2 (M+H) ⁺ .

Stereochemistry was assigned based on use of (2R)-2-methylpyrrolidine in step 1 of the synthesis of Intermediate 4 and (S)-(-)-2-methyl-2-propanesulfinamide in step 2.

Intermediate 10: (S, S)-2-methyl-N-[(1 R)-1 -{6-[methyl(propan-2-yl)am ino]-1 -oxo-

2,3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}propyl]propane-2-sulfinamide

Step 1 : 6-[methyl(propan-2-yl)amino]-1 -oxo-2,3-dihydro-1 /-/-pyrrolo[3,4- c]pyridine-4-carbonitrile A mixture of 4-chloro-6-[methyl(propan-2-yl)amino]-2,3-dihydro-1/-/-pyrro lo[3,4- c]pyridin-1 -one (9d) (706 mg, 2.95 mmol), Zn(CN)2 (519 mg, 4.42 mmol), DMF (10 mL) and Pd(PPh3)4 (170 mg, 0.147 mmol) was heated to 140 °C in the microwave for 30 min. The reaction was diluted with DCM and filtered. The filter cake was washed with DCM. The filtrate was concentrated in vacuo to provide the title compound (10a) as a yellow solid (544 mg, 80% yield) which was used without further purification. ¹ H NMR (400 MHz, DMSO-cfe) 9.01 (s, 1 H), 7.13 (s, 1 H), 4.80 (td, J = 6.7, 13.4 Hz, 1 H), 4.47 (s, 2H), 2.89 (s, 3H), 1.15 (d, J = 6.7 Hz, 6H); m/z (APCI+) for (C12H14N4O), 231.2 (M+H) ⁺ .

Step 2: 6-[methyl(propan-2-yl)amino]-4-propanoyl-2,3-dihydro-1 /-/-pyrrolo[3,4- c]pyridin-1-one (10b)

A suspension of 6-[methyl(propan-2-yl)amino]-1 -oxo-2,3-dihydro-1 /-/-pyrrolo[3,4- c]pyridine-4-carbonitrile (10a) (511 mg, 2.22 mmol) in THF (20.0 mL) was cooled to 0 °C and then treated with a solution of ethylmagnesium bromide (3.0 M in Et20, 7.40 mL, 22.2 mmol). The mixture was stirred at RT for 1 h. LCMS analysis showed consumption of the starting material. The reaction was cooled to 0 °C and quenched by addition of saturated NH4CI (60 mL) and then extracted with DCM (2 x 80 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (24 g SiC>2, 0-100% EtOAc/heptane) to provide the title compound (10b) (125 mg, 22% yield) as a yellow foam, m/z (APCI+) for (C14H19N3O2), 262.2 (M+H) ⁺ .

Step 3: Intermediate 10

To a solution of 6-[methyl(propan-2-yl)amino]-4-propanoyl-2,3-dihydro-1 /-/- pyrrolo[3,4-c]pyridin-1 -one (10b) (124 mg, 0.475 mmol) in THF (6.0 mL) were added (S)- (-)-2-methyl-2-propanesulfinamide (92 mg, 0.759 mmol) and Ti(0Et)4 (433 mg, 1.90 mmol). The mixture was stirred at reflux for 42 h. LCMS analysis showed consumption of the starting material. The mixture was cooled to -78 °C and then treated dropwise with a solution of L-selectride (1.0 M in THF, 1.9 mL, 1.90 mmol). The mixture was stirred at - 78 °C for 4 h. The mixture was quenched with MeOH, diluted with brine (30 mL), and extracted with DCM (20 x 2 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (12 g SiC>2, 0-10% MeOH/DCM) to provide Intermediate 10 (72 mg, 41 % yield) as a yellow solid. ¹ H NMR (400 MHz, DMSO-de) 5 8.67 (s, 1 H), 6.64 (s, 1 H), 5.25 (d, J = 6.7 Hz, 1 H), 4.94 (quin, J = 6.7 Hz, 1 H), 4.34 (s, 2H), 4.21 (q, J = 6.7 Hz, 1 H), 2.84 (s, 3H), 1 .98 - 1 .80 (m, 2H), 1.13 (dd, J = 3.6, 6.7 Hz, 6H), 1 .06 (s, 9H), 0.83 (t, J = 7.3 Hz, 3H). m/z (APCI+) for (C18H30N4O2S), 367.2 (M+H) ⁺ . Stereochemistry was assigned based on use of (S)-(-)- 2-methyl-2-propanesulfinamide in step 2.

Intermediate 13: (S,S)-2-methyl-/V-[(1 R)-1 -{6-[methyl(propan-2-yl)amino]-1 - oxo-2, 3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}ethyl]propane-2-sulfinamide

Step 1 : 4-acetyl-6-(isopropyl(methyl)amino)-2,3-dihydro-1 H-pyrrolo[3,4-c]pyridin- 1 -one

Methylmagnesium bromide (9.11 g, 76.4 mmol, 25.9 mL, 3.0 M) was added to a solution of 6-(isopropyl(methyl)amino)-1 -oxo-2, 3-dihydro-1 /-/-pyrrolo[3,4-c]pyridine-4- carbonitrile (Intermediate 10a) (1.76 g, 7.64 mmol) in THF (70 mL, c=0.11 M) at 0 °C . The mixture was stirred at this temperature for 10 min and then allowed to warm to RT and allowed to stir for 3 h. The mixture was then quenched with 2 M HCI (7 mL) dropwise at RT and allowed to stir for 30 min. A saturated solution of NaHCOs (70 mL) was added to neutralize the solution and it was extracted with DCM (3 x 100 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The resulting residue was purified by flash chromatography (40 g SiC>2, 10-40% EtOAc/heptane) to provide the title compound (13a) as a yellow solid (553 mg, 29% yield). ¹ H NMR (400 MHz, DMSO-d6) 5 8.82 (s, 1 H) 7.06 (s, 1 H) 4.88 (br. d, J = 6.72 Hz, 1 H) 4.49 (s, 2H) 2.94 (s, 3H) 2.61 (s, 3H) 1 .19 (d, J = 6.60 Hz, 6H); m/z (APCI+) for (C13H17N3O2), 248.2 (M+H) ⁺ .

Step 2: Intermediate 13

A mixture of 4-acetyl-6-[methyl(propan-2-yl)amino]-2,3-dihydro-1/-/-pyrro lo[3,4- c]pyridin-1 -one (13a) (8.10 g, 32.75 mmol ), (S)-fe/t-butylsulfinamide (7.94 g, 65.5 mmol) in Ti(0Et)4 (29.9 g, 131 mmol) in THF (100 mL) under N2 was heated at 90 °C for 72 h and the reaction was monitored by LCMS. The reaction was then cooled to 0 °C and L- selectride (1 M in THF, 131 mmol, 131 mL) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 3 h. The mixture was quenched with saturated NH4CI (200 mL) and brine (200 mL) at 0-5 °C. The suspension was filtered through a pad of Celite® and the filter cake was washed with DCM (500 mL). The filtrate layers were separated, and the aqueous layer was extracted with DCM (2 x 100 mL). The combined organic layers were washed with brine (250 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography (SiC>2, 0-10% MeOH/DCM) to provide Intermediate 13 as a yellow solid (10 g, 87%). ¹ H NMR (400 MHz, CDCI3) 5 6.83 (br. s, 1 H), 6.75 (s, 1 H), 4.89 - 4.76 (m, 1 H), 4.48 - 4.39 (m, 1 H), 4.37 - 4.26 (m, 3H), 2.83 (s, 3H), 1.55 (d, J = 6.6 Hz, 3H), 1.19 - 1.07 (m, 15H). m/z (APCI+) for (C17H28N4O2S), 353.2 (M+H) ⁺ . Stereochemistry was assigned based on use of (S)-ferf-butylsulfinamide in step 1.

Intermediate 14: (S _; R)-2-methyl-A/-[(1 S)-1 -{6-[(2R)-2-methylpyrrolidin-1-yl]-1- oxo-2, 3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}ethyl]propane-2-sulfinamide

A mixture of 4-acetyl-6-[(2R)-2-methylpyrrolidin-1 -yl]-2,3-dihydro-1 /-/-pyrrolo[3,4- c]pyridin-1 -one (4f) (4000 mg, 15.43 mmol), (R)-2-methylpropane-2-sulfinamide (2240 mg, 18.5 mmol) in Ti(0Et)4 (20 mL) was stirred at 100 °C for 20 h under N2. LCMS analysis showed consumption of the starting material. The yellow solution was cooled to 0 °C and was diluted with THF (50 mL). L-Selectride (38.6 mmol, 38.6 mL, 1 M in THF) was added dropwise at 0 °C. The mixture was then stirred at 10 °C for 20 h. LCMS showed starting material remaining. Additional L-Selectride (38.6mmol, 38.6 mL 1 M in THF) was added dropwise at 0 °C and the mixture was stirred at 10 °C for an additional 2 h. TLC analysis showed consumption of the starting material. The mixture was quenched with saturated aqueous NH4CI (200 mL) at 0-5 °C. The suspension was filtered through a pad of Celite® and the cake was washed with EtOAc (2 x 50 mL). The filtrate layers were separated, and the aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated to provide a residue. The residue was first purified by flash chromatography (SiO2, EtOAc/MeOH = 10:1 ) and then further purified by prep HPLC (ACSSH-CA; Method column: YMC Triart C18 250*50mm*7pm; water (0.05% ammonia hydroxide v/v)-ACN to provide Intermediate 14 (2.5 g, 44%) as an off-white solid. ¹ H NMR (400 MHz, CDCI3) 5 6.70 (s, 1 H), 6.47 (br. s, 1 H), 4.57 - 4.34 (m, 4H), 4.20 (br. t, J = 5.9 Hz, 1 H), 3.64 - 3.53 (m, 1 H), 3.47 - 3.35 (m, 1 H), 2.20 - 1 .99 (m, 3H), 1 .77 (br. dd, J = 2.5, 4.7 Hz, 1 H), 1 .64 (d, J = 6.5 Hz, 3H), 1.28 (d, J = 6.2 Hz, 3H), 1.22 (s, 9H). m/z (ESI) for (C18H28N4O2S), 365.1 (M+H) ⁺ . Stereochemistry was assigned based on use of (R)-tert-butylsulfinamide.

Example 1

4-[(1 R)-1 -aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2 ,1 -c][1 ,2,4]triazol-3- y l]py rid in-2-y l}-6-[(2R)-2-m ethy Ipyrrol id in-1 -y l]-2 , 3-d ihydro-1 /-/-pyrrolo[3,4-c]pyrid in-1 - one

Step 1 : (S,S)-2-methyl-N-[(1 R)-1 -(2-{6-[(5S)-5-methyl-6,7-dihydro-5H-pyrrolo[2,1- c][1 ,2 , 4]triazol-3-yl]py rid in-2-yl}-6-[(2R)-2-m ethy Ipyrrol idin-1 -y l]-1 -oxo-2,3-dihydro-1 H- pyrrolo[3,4-c]pyridin-4-yl)propyl]propane-2-sulfinamide

A mixture of (S,S)-2-methyl-A/-[(1 R)-1-{6-[(2R)-2-methylpyrrolidin-1-yl]-1 -oxo-2,3- dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}propyl]propane-2-sulfinamide (Intermediate 5) (63.0 mg, 0.17 mmol), (5S)-3-(6-bromopyridin-2-yl)-5-methyl-6,7-dihydro-5/-/- pyrrolo[2,1 -c][1 ,2,4]triazole (Intermediate 2) (147 mg, 0.528 mmol), K3PO4 (336 mg, 1.59 mmol), Pd2(dba)s (30.4 mg, 0.0528 mmol) and XantPhos (61.1 mg, 0.106 mmol) in 1 ,4-dioxane (4.5 mL, c=0.1 M) was heated at 100 °C (block temp) in 40 mL vial (capped) for 18 h. The mixture was filtered through Celite®, washed with DCM. The filtrate was concentrated in vacuo and the crude compound was purified by flash chromatography (SiC>2, 0-10% MeOH/DCM) to provide the title compound as pale yellow color foam (194 mg, 64% yield), m/z (APCI+) for (C29H40N8O2S), 577.3 (M+H) ⁺ .

Step 2: Example 1

A solution of 4 N HCI in 1 ,4-dioxane (1 .01 mmol, 0.252 mL, 4 M) was added to a suspension of (S,S)-2-methyl-N-[(1 R)-1-(2-{6-[(5S)-5-methyl-6,7-dihydro-5H-pyrrolo[2,1- c][1 ,2 , 4]triazol-3-yl]py rid in-2-yl}-6-[(2R)-2-m ethylpyrrol idin-1 -y l]-1 -oxo-2,3-dihydro-1 H- pyrrolo[3,4-c]pyridin-4-yl)propyl]propane-2-sulfinamide (194 mg, 0.336 mmol) in MeOH (12 mL). The resulting mixture was stirred at RT for 2 h. The volatiles were removed under reduced pressure. The crude product was purified by Chiral SFC (Phenomenex Lux Cellulose-1 4.6 x 100mm 3pm column 30% MeOH + 10 mM NH3 in CO2 @ 120 bar, 4 mL/min) to provide Example 1 (146 mg, 92% yield, >99% de) as pale yellow color solid. ¹ H NMR (600 MHz, DMSO-de) 5 8.48 (d, J = 8.3 Hz, 1 H), 8.01 (t, J = 8.0 Hz, 1 H), 7.91 (d, J = 7.7 Hz, 1 H), 6.67 (s, 1 H), 5.22 - 5.03 (m, 3H), 4.33 - 4.21 (m, 2H), 3.60 - 3.50 (m, 1 H), 3.41 - 3.32 (m, 1 H), 3.06 - 2.98 (m, 1 H), 2.97 - 2.89 (m, 1 H), 2.88 - 2.76 (m, 1 H), 2.37 - 2.27 (m, 1 H), 2.09 - 1 .86 (m, 5H), 1 .69 - 1 .62 (m, 1 H), 1 .44 (d, J = 6.4 Hz, 3H), 1 .15 (d, J = 6.2 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H); m/z (APCI+) for (C26H32N8O), 473.2 (M+H) ⁺ ; [D]D22 = +37.0° (c=0.1 M, MeOH).

Example 2 4-[(1R)-1-aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5/-/-py rrolo[2,1-c][1 ,2,4]triazol-3- y l]py rid in-2-y l}-6-[(2R)-2-m ethy Ipyrrol id in-1 -y l]-2 , 3-d ihydro-1 /-/-pyrrolo[3,4-c]pyrid in-1 - one

Step 1 : (S,S)-N-[(1 R)-1 -(2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2, 1 - c][1 ,2 , 4]triazol-3-yl]py rid in-2-yl}-6-[(2R)-2-m ethy Ipyrrol idin-1 -y l]-1 -oxo-2,3-dihydro-1 H- pyrrolo[3,4-c]pyridin-4-yl)ethyl]-2-methylpro pane-2 -sulfinamide

To a mixture of (S,S)-2-methyl-N-[(1R)-1-{6-[(2R)-2-methylpyrrolidin-1-yl]-1 -oxo- 2,3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}ethyl]propane-2-sulfinamide (Intermediate 4) (50 mg, 0.14 mmol), (5S)-3-(6-bromopyridin-2-yl)-5-ethyl-6,7-dihydro-5/-/-pyrrol o[2,1- c][1 ,2,4]triazole (Intermediate 3) (42.2 mg, 0.144 mmol), and K3PO4 (87.4 mg, 0.412 mmol) in 1 ,4-dioxane (3 mL) was added Pd2(dba)s (12.6 mg, 0.014 mmol) andXantPhos (15.9 mg, 0.027 mmol) under N2. After addition, the mixture was bubbled with N2 for 2 min. The resulting mixture was sealed and stirred at 85 °C for 18 h. The reaction mixture was concentrated in vacuo, and the residue was purified by flash chromatography (S iC>2, 10% EtOAc/MeOH) to provide the title compound (60 mg, 76% yield) as a yellow solid. ¹ H NMR (400 MHz, CDCI3) 58.70 (d, J = 8.2 Hz, 1H), 8.18 (d, J = 7.4 Hz, 1H), 7.93 (t, J = 8.0 Hz, 1H), 6.76 (s, 1H), 5.11 -5.02 (m, 2H), 4.99 - 4.93 (m, 1H), 4.61 -4.49 (m, 2H), 4.34-4.27 (m, 1H), 3.67-3.57 (m, 1H), 3.46-3.35 (m, 1H), 3.11 -3.01 (m, 3H), 2.65 (td, J = 3.7, 7.9 Hz, 1H), 2.19-2.08 (m, 3H), 2.07 (s, 1H), 1.86-1.79 (m, 1H), 1.79 (br. d, J = 2.3 Hz, 1 H), 1.69 (d, J = 6.4 Hz, 3H), 1.31 -1.28 (m, 3H), 1.22 (s, 9H), 1.02 (t, J = 7.5 Hz, 3H); m/z (ESI+) for (C30H40N8O2S), 577.5 (M+H) ⁺ ; [a] D22 = +85.3° (c=0.1 M, MeOH).

Step 2: Example 2

To a solution of (S,S)-N-[(1 R)-1 -(2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2, 1 - c][1 ,2 , 4]triazol-3-yl]py rid in-2-yl}-6-[(2R)-2-m ethy Ipyrrol idin-1 -y l]-1 -oxo-2,3-dihydro-1 H- pyrrolo[3,4-c]pyridin-4-yl)ethyl]-2-methylpro pane-2-sulfinamide (60 mg, 0.10 mmol) in EtOAc (5mL) was added dropwise 4 M HCI in EtOAc (3 mL) at 0 °C. The mixture was stirred at 20 °C for 1 h. The reaction mixture was concentrated in vacuo. To the residue was added EtOAc (10 mL) and water (10 mL). The aqueous layer was basified with saturated NaHCOs and extracted with DCM (10 mL x 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was lyophilized for 16 h to provide Example 2 (47 mg, 95%) as pale yellow color solid. ¹ H NMR (400 MHz, DMSO-de) 5 8.59 (d, J = 8.3 Hz, 1 H), 8.09 - 8.02 (m, 1 H), 8.02 - 7.97 (m, 1 H), 6.58 (s, 1 H), 5.33 (d, J = 17.1 Hz, 1 H), 5.10 (d, J = 17.1 Hz, 1 H), 4.99 (br. t, J = 6.1 Hz, 1 H), 4.29 - 4.19 (m, 1 H), 4.12 (q, J = 6.9 Hz, 1 H), 3.55 (br. t, J = 8.0 Hz, 1 H), 3.04 - 2.85 (m, 3H), 2.58 - 2.55 (m, 1 H), 2.1 1 - 1 .96 (m, 4H), 1 .79 - 1 .66 (m, 2H), 1 .36 (d, J = 6.5 Hz, 3H), 1 .24 - 1 .20 (m, 3H), 0.93 (t, J = 7.4 Hz, 3H); m/z (ESI+) for (C26H32N8O), 473.4 (M+H) ⁺ .

Example 3

4-[(1 S)-1 -aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2, 1 -c][1 ,2,4]triazol-3- y l]py rid in-2-y l}-6-[(2R)-2-m ethy Ipyrrol id in-1 -y l]-2 , 3-d ihydro-1 /-/-pyrrolo[3,4-c]pyrid in-1 - one

Step 1 : (S,R)-N-[(1 S)-1 -(2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2, 1 - c][1 ,2 , 4]triazol-3-yl]py rid in-2-yl}-6-[(2R)-2-m ethy Ipyrrol idin-1 -y l]-1 -oxo-2,3-dihydro-1 H- pyrrolo[3,4-c]pyridin-4-yl)ethyl]-2-methylpro pane-2 -sulfinam ide

To a suspension of (S _; R)-2-methyl-/V-[(1 S)-1 -{6-[(2R)-2-methylpyrrolidin-1-yl]-1- oxo-2, 3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}ethyl]propane-2-sulfinamide (Intermediate 14) (2000 mg, 5.487 mmol), (5S)-3-(6-bromopyridin-2-yl)-5-ethyl-6,7-dihydro-5/-/- pyrrolo[2,1 -c][1 ,2,4]triazole (Intermediate 3) (1610 mg, 5.49 mmol) and KsP04 (3490 mg, 16.5 mmol) in 1 ,4-dioxane (30 mL), Pd2(dba)s (502 mg, 0.549 mmol) and XantPhos (635 mg, 1.10 mmol) were added under N2. After addition, the mixture was bubbled with Argon for 2 min. The resulting mixture was sealed and allowed to stir at 85 °C for 18 h. TLC analysis showed consumption of the starting material. The mixture was diluted with H2O (100 mL) with stirring and the resultant suspension was filtered. The aqueous filtrate was separated. The solid was washed with DCM (100 mL). The organic layer was dried over Na2SO4, filtered and concentrated to provide a yellow residue (3 g) which was purified by flash chromatography (SiC>2, 0-10% MeOH in EtOAc) to provide a yellow solid. The solid was dissolved in DCM (50 mL) and silica-SH (4 g) was added. The yellow mixture was refluxed for 20 min. The mixture was filtered, and the filter cake was washed with DCM/MeOH (10:1 ). The filtrate was concentrated. The treatment with silica-SH was repeated three additional times to provide the title compound (2.3 g, 72.7%) as a yellow solid. ¹ H NMR (400 MHz, CDCI3) 5 8.71 (dd, J = 0.7, 8.4 Hz, 1 H), 8.18 (dd, J = 0.7, 7.6 Hz, 1 H), 7.97 - 7.87 (m, 1 H), 6.76 (s, 1 H), 5.19 - 5.11 (m, 1 H), 5.03 - 4.94 (m, 2H), 4.65 - 4.57 (m, 1 H), 4.57 - 4.51 (m, 1 H), 4.24 (br t, J = 6.1 Hz, 1 H), 3.66 - 3.56 (m, 1 H), 3.48 - 3.39 (m, 1 H), 3.13 - 2.98 (m, 3H), 2.71 - 2.59 (m, 1 H), 2.21 - 2.03 (m, 4H), 1.87 - 1.75 (m, 2H), 1 .68 (d, J = 6.5 Hz, 4H), 1 .30 (d, J = 6.2 Hz, 3H), 1 .24 (s, 9H), 1 .02 (t, J = 7.5 Hz, 3H). m/z (ESI) for (C30H40N8O2S), 577.5 (M+H) ⁺ .

Step 2: Example 3

To a solution of (S,R)-N-[(1 S)-1-(2-{6-[(5S)-5-ethyl-6,7-dihydro-5H- pyrrolo[2, 1 -c][1 , 2 , 4]triazol-3-y l]py rid in-2-y l}-6-[(2R)-2-m ethy Ipy rrol id in-1 -y l]-1 -oxo-2,3- dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl)ethyl]-2-methylpro pane-2 -sulfinam ide (2300 mg, 3.988 mmol) in EtOAc (10 mL) was added 4M HCI in EtOAc (20 mL) at 0 °C. After addition, the mixture was stirred at 15 °C for 1 h. The reaction was monitored by LCMS. The resulting yellow suspension was concentrated to provide a residue which was dissolved in H2O (30 mL) and extracted with EtOAc (25 mL). The aqueous layer was basified with a solution of saturated NaHCOs to pH ~ 8 and extracted with DCM (3 x 35 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to provide a solid to which DCM (50 mL) was added. The solution was filtered through glass microfiber filter GF/F (~0.7 pm) four times. The filtrate was concentrated to provide a yellow solid which was purified by prep. HPLC (Column: Agela DuraShell C18 150*40mm*5pm, water (0.05% ammonia hydroxide v/v)-CAN, Gradient Time 12 min, Flow Rate 25 mL/min). This provided Example 3 (1.5 g, 79.6%) as a yellow solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) 6 8.55 (d, J = 8.0 Hz, 1 H), 8.08 - 7.90 (m, 2H), 6.51 (s, 1 H), 5.26 (d, J = 16.8 Hz, 1 H), 5.08 - 4.92 (m, 2H), 4.23 (br. t, J = 5.8 Hz, 1 H), 4.09 (q, J = 6.5 Hz, 1 H), 3.53 (br. t, J = 7.5 Hz, 1 H), 3.30 - 3.23 (m, 1 H), 3.07 - 2.83 (m, 3H), 2.62 - 2.53 (m, 1 H), 2.16 - 1 .85 (m, 6H), 1 .77 - 1 .64 (m, 2H), 1 .33 (d, J = 6.5 Hz, 3H), 1 .21 (d, J = 6.0 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H). m/z (ESI) for (C26H32N8O), 473.4 (M+H) ⁺ .

Example 4

4-[(1R)-1-aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-p yrrolo[2,1-c][1 ,2,4]triazol-3- yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-2,3-dihydro-1/ -/-pyrrolo[3,4-c]pyridin-1-one

Step 1 : (S,S)-N-[(1 R)-1 -(2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2, 1 - c][1 ,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino] -1-oxo-2,3-dihydro-1/-/- pyrrolo[3,4-c]pyridin-4-yl)ethyl]-2-methylpropane -2-sulfinamide

A solution of (S _; S)-2-methyl-N-[(1 R)-1 -{6-[methyl(propan-2-yl)amino]-1 -oxo-2,3- dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}ethyl]propane-2-sulfinamide (Intermediate 13) (627 mg, 1.78 mmol), (5S)-3-(6-bromopyridin-2-yl)-5-ethyl-6,7-dihydro-5/-/-pyrrol o[2,1- c][1 ,2,4]triazole (Intermediate 3) (521 mg, 1.78 mmol), K3PO4 (1.13 g, 5.34 mmol), Pd2(dba)3 (102 mg, 0.178 mmol) and XantPhos (206 mg, 0.356 mmol) in 1 ,4- dioxane (17.8 mL, c=0.1 M) was heated at 100 °C in a 100 mL flask with condenser under N2 for 18 h. The mixture was cooled to RT, filtered and washed with DCM (20 mL). The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (24 g, S iC>2, 0-10% MeOH/DCM) to provide the title compound as a paleyellow color solid (808 mg, 80%). m/z (APCI+) for (C29H40N8O2S), 565.3 (M+H) ⁺ .

Step 2: Example 4

A 4 N solution of HCI in 1 ,4-dioxane (1 .07 mL, 4.29 mmol) was added to a solution of (S,S)-N-[(1R)-1-(2-{6-[(5S)-5-ethyl-6,7-dihydro-5/-/-pyrrolo [2,1 -c][1 ,2,4]triazol-3- yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-1 -oxo-2, 3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin- 4-yl)ethyl]-2-methylpropane-2-sulfinamide (808 mg, 1 .43 mmol) in MeOH (14.3 mL, c=0.1 M). The mixture was stirred at RT for 2 hr. The volatile materials were removed under reduced pressure. The crude product was purified by Chiral SFC (Phenomenex Lux Cellulose-1 21 x 250 mm column, 30% MeOH + 10mM NH3 in CO2 held at 120 bar, 100 mL/min) to provide Example 4 (345 mg, 52% yield, >99% de, >99% pure) as pale yellow color solid. ¹ H NMR (400 MHz, DMSO-de) 5 8.56 (dd, J = 1.0, 8.1 Hz, 1 H), 8.07 - 8.01 (m, 1 H), 8.00 - 7.93 (m, 1 H), 6.78 (s, 1 H), 5.34 - 5.28 (m, 1 H), 5.17 - 5.10 (m, 1 H), 5.02 - 4.95 (m, 1 H), 4.89 (td, J = 6.7, 13.4 Hz, 1 H), 4.18 (q, J = 6.7 Hz, 1 H), 3.01 - 2.94 (m, 2H), 2.93 (s, 3H), 2.62 - 2.54 (m, 2H), 2.09 - 1 .97 (m, 1 H), 1.84 - 1 .72 (m, 1 H), 1 .41 (d, J = 6.6 Hz, 3H), 1.20 (d, J = 6.6 Hz, 6H), 0.92 (t, J = 7.5 Hz, 3H); m/z (APCI+) for (C25H32N8O), 461.3 (M+H) ⁺ . [a]D ₂ 2 = +120.5° (c=0.1 M, MeOH). Example 5

4-[(1 R)-1 -aminopropyl]-2-{3-[(5S)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2 ,1 -c][1 ,2,4]triazol-3- yl]phenyl}-6-[methyl(propan-2-yl)amino]-2,3-dihydro-1 /-/-pyrrolo[3,4-c]pyridin-1 -one

Step 1 : (S _; S)-2-methyl-N-[(1 R)-1 -(2-{3-[(5S)-5-methyl-6,7-dihydro-5H-pyrrolo[2,1- c][1 ,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]-1 -oxo-2,3-dihydro-1 H- pyrrolo[3,4-c]pyridin-4-yl)propyl]propane-2-sulfinamide

A mixture of (S _; S)-2-methyl-A/-(1 -{6-[methyl(propan-2-yl)amino]-1 -oxo-2,3- dihydro-1 /-/-pyrrolo[3,4-c]pyridin-4-yl}propyl)propane-2-sulfinamide (Intermediate 10) (63 mg, 0.17 mmol), (5S)-3-(3-bromophenyl)-5-methyl-6,7-dihydro-5/-/-pyrrolo[2,1 - c][1 ,2,4]triazole (Intermediate 2) (48 mg, 0.172 mmol), K3PO4 (109 mg, 0.516 mmol), Pd2(dba)3 (9.88 mg, 0.0172 mmol) and XantPhos (19.9 mg, 0.0344 mmol) in 1 ,4- dioxane (4.5 mL, c=0.1 M) was heated at 100 °C in a 40 mL vial (capped) for 18 h. The volatiles were removed under reduced pressure. The residue was purified by flash chromatography (12 g, S iC>2, 0-10% MeOH/DCM) to provide the title compound as a paleyellow color foam (74.0 mg, 76%). m/z (APCI+) for (C29H40N8O2S), 565.3 (M+H) ⁺ .

Step 2: Example 5

A 4 N solution of HCI in 1 ,4-dioxane (0.164 mL, 0.655 mmol) was added to a solution of (S,S)-2-methyl-A/-[(1 R)-1 -(2-{3-[(5S)-5-methyl-6,7-dihydro-5H-pyrrolo[2,1- c][1 ,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]-1 -oxo-2,3-dihydro-1 H- pyrrolo[3,4-c]pyridin-4-yl)propyl]propane-2-sulfinamide (74 mg, 0.13 mmol) in MeOH (5.0 mL, c=0.026 M). The mixture was stirred at RT for 2 h. The volatiles were removed under reduced pressure. The crude product was purified by Chiral SFC (Phenomenex Lux Cellulose-1 4.6 x 100mm 3pm column 5-60% MeOH + 10 mM NH3 in CO2 ramping over 3.0 minutes @ 120 bar, 4 mL/min) to provide Example 5 (11.6 mg, 19% yield, >99% de, >95% pure) as pale yellow color solid. ¹ H NMR (600 MHz, DMSO-cfe) 5 8.54 (d, J = 8.3 Hz, 1 H), 8.35 (br. s, 2H), 8.07 (t, J = 8.0 Hz, 1 H), 8.00 - 7.90 (m, 1 H), 6.91 (d, J = 0.9 Hz, 1 H), 5.27 - 5.07 (m, 3H), 5.01 (br. s, 1 H), 4.37 (br. s, 1 H), 3.14 - 2.97 (m, 2H), 2.94 (d, J = 1 .5 Hz, 3H), 2.92 - 2.85 (m, 1 H), 2.42 - 2.35 (m, 1 H), 2.06 - 1 .91 (m, 2H), 1 .50 (d, J = 6.1 Hz, 3H), 1.19 (d, J = 6.6 Hz, 3H), 1.15 (d, J = 6.6 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H); m/z (APCI+) for (C25H32N8O), 461.3 (M+H) ⁺ . [a]D ₂₂ = +75.9° (c=0.2 M, MeOH).

Example 6

In Vitro Growth Inhibition Study by the Combination of HPK1 Inhibitor PF-07265028 and Anti-PD1 Antibody Sasanlimab

The purpose of this example was to evaluate the effects of the HPK1 inhibitor PF- 07265028 on the ability of tumor-reactive T cells to kill human A375 tumor cells, and to evaluate the potential of PF-07265028 with or without an anti-PD1 antibody of the present embodiments (sasanlimab) as a treatment option for cancer.

Materials and Methods

Cell Culture:

To generate tumor-reactive T cells, CD4 or CD8 T cells were isolated from PBMCs following the EasySep Human T cell isolation kit (obtained from StemCell Technologies) protocol, and mixed with mitomycin treated A375 cells (10 pg/mL Mitomycin C for 2 hours; mitomycin C is obtained from StemCell Technologies) in X-VIVO™ 15 medium (obtained from Lonza Walkersville, Inc.) with 10% human serum AB (obtained from Fisher Scientific). After 10 minutes, 20 ng/mL recombinant human IL-2 (IS) and 10 ng/mL recombinant human IL-7 (obtained from Miltenyi Biotec) were added to expand activated T cells, and expansion was continued in G-Rex culture vessels for 21 days. The resulting cells were cryopreserved in CryoStor CS10 (obtained from StemCell).

A375 human melanoma cells were passaged in Gibco Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and 1 % penicillin/streptomycin. Prior to co-culture assays, the A375 cell line was transduced with Incucyte NucLight Green Lentivirus (nucGFP) (obtained from Sartorius) and selected by 1 pg/mL puromycin (obtained from Sigma).

Tumor Killing Assay:

A375 cells expressing nucGFP were seeded at 5,000 to 10,000 cells per well in 100 pL XVIVO™ 15 with 10% human serum AB (assay medium) in an ImageLock 96- well plate. Cells were cultured for 24 hours in a humidified incubator at 37°C and 5% CO2. The following day, frozen CD4 and CD8 cells were thawed in 37°C assay medium and rested for 2 hours. Meanwhile the media on the previously plated A375 cells was replaced with 50 pL PBS containing 3x Annexin V-Red staining reagent and 3 mM CaCl2. About 8 to 300 nM concentration of PF-07265028 and/or 2 pg/mL sasanlimab was added to each well in 50 pL of assay medium, and T cells were added in 50 pL of assay medium as well. Equal amounts of CD4 and CD8 T cells were mixed, and then added to each well. Wells contained either 2, 4, 5, 10, or 20 T cells for every tumor cell depending on the specified ratio. Plates were then transferred to the Incucyte S3 imager and monitored for 4 days.

Data Analysis:

The Incucyte S3 imager monitors A375 proliferation through the nuclear GFP and apoptosis through the Annexin V-Red stain and reports the average number of green and red objects per field of view.

The percent of A375 clearance was calculated using the formula: green object count with T cells, with compound

1 - - : - X 100 green object count without T cells, with compound

The percent of A375 apoptosis was calculated using the formula: red object count with T ceils, with compound red object count without T cells, with comopund - - - X 100 green object count with T ceils, with compound green object count without T cells, with compound

Results:

Tumor-reactive T cells were generated against the A375 cell line and then cocultured with A375 tumor cells expressing nucGFP in the presence of an apoptosis marker (Annexin V Red, obtained from Sartorius). Through imaging the frequency of GFP-positive cells, the expansion and loss of A375 cells was monitored over the course of four days. A375-nucGFP cells were counted by fluorescence microscopy examining the number of green nuclei. Cells were treated with 111 nM PF-07265028 alone, 2 pg/mL sasanlimab alone, or the combination of those agents. The controls were either A375 cell growth without T cells, or mock-treated T cells (in DMSO). The effect of PF-07265028 alone and in combination with sasanlimab on the growth of the A375 tumor cells is shown in FIG. 1. A ratio of six T cells were plated for each tumor cell. Each value is the average of six replicates with error bars representing the SEM.

Calculation of % A375 clearance is described in the methods and reports the reduction in A375-nucGFP cells by 96 hours. Increasing amounts of PF-07265028 were evaluated either alone or in combination of 2 pg/mL sasanlimab. FIG. 2 shows the dose response of PF-07265028 alone and in combination with sasanlimab on the growth of the A375 tumor cells in a T cell co-culture. The controls show either A375 cell growth with DMSO treatment (Mock) or DMSO with sasanlimab. Each value is the average of six replicates with error bars representing the SEM.

To assess the potential combination benefit of PF-07265028 with sasanlimab, cocultures of T cells and tumor cells were evaluated in the presence of sasanlimab and increasing concentrations of PF-07265028. As described above, tumor-reactive T cells were co-cultured with A375-nucGFP cells in the presence of an apoptosis marker (Annexin V Red). FIG. 3 shows the effect of PF-07265028 alone and in combination with sasanlimab on the growth of the A375 apoptotic tumor cells in a T cell co-culture. In cocultures of T cells and tumor cells, PF-07265028 increases the number of apoptotic tumor cells. Apoptotic A375-nucGFP tumor cells were imaged by an Annexin V-Red stain, and the %A375 apoptotic cells after 96 hours of co-culture was calculated as described in the methods. Increasing amounts of PF-07265028 was evaluated either alone or in combination of 2 pg/mL sasanlimab. The controls show either A375 cell growth with DMSO treatment (Mock) or DMSO with sasanlimab. Each value is the average of six replicates with error bars describing the SEM.

In co-cultures of T cells and tumor cells, PF-07265028 increases the amount of IFNy produced by T cells. After 96 hours of co-culture, supernatants were collected and analyzed by MSD quantitative immunoassay. Increasing amounts of PF-07265028 were evaluated either alone or in combination of 2 pg/mL sasanlimab. FIG. 4 shows the effect of PF-07265028 alone and in combination with sasanlimab on IFNy production in a T cell co-culture with tumor cells. The controls show either A375 cell growth with DMSO treatment (Mock) or DMSO with sasanlimab. Each value is the average of six replicates with error bars representing the SEM.

Control of Tumor Cell Growth by T Cells and PF-07265028

It is observed that the addition of 300 nM PF-07265028 resulted in 33.9% A375 clearance after 96 hours whereas mock-treated co-cultures only cleared 9.4% of A375 (Table 1 , FIG. 1). The clearance of A375 cells was due, in part, to increased A375 apoptosis as measured by the Annexin V Red stain. While mock-treated T cells led to 22.2% A375 apoptosis after 96 hours, addition of 300 nM PF-07265028 resulted in 31 .9% (Table 1). Consistent with an increase in T cell activity, an increase in IFNy was observed, increasing from 8,165 pg/mL with mock-treated T cells to 15,651 pg/mL following 300 nM PF-07265028 (Table 1). An anti-tumor dose-response was observed following incubation with PF-07265028 with the values listed in Table 1, and plotted in FIGS. 2, 3, and 4.

Table 1: Enhanced Tumor Cell Control by T cells with PF-07265028 at 96 Hours

Tabulated above are values plotted in FIGS. 2, 3, and 4 and represent the average and SEM of six replicates. The % A375 clearance and apoptosis are based on live-cell imaging of A375 tumor cells and are calculated as described in the methods. IFNy was measured by MSD immunoassay.

PF-07265028 Effect with and Sasanlimab in Controlling Tumor Cell Growth

Sasanlimab alone increased the clearance of A375 cells at 96 hours from 9.4% to 29.5% (Table 2, FIG. 1). Addition of 300 nM PF-07265028 further increased A375 clearance to 52.6%. For % A375 apoptosis, there was an increase from 30.2% to 45.6% comparing sasanlimab alone to the combination of sasanlimab with 300 nM PF-07265028 (Table 2, FIG. 3). Consistent with this, IFNy increased from 14,428 pg/mL to 26,082 pg/mL (Table 2, FIG. 4). PF-07265028 dose responses are tabulated in Table 1 and graphed in FIG. 2, FIG. 3, and FIG. 4, and demonstrate a combination benefit across 8 to 300 nM PF-07265028.

Table 2: Combination Benefit of PF-07265028 and Sasanlimab at 96 Hours

Tabulated above are values plotted in FIG. 2 and FIG. 3 and represent the average and SEM of six replicates. Sasanlimab was added at 2 pg/mL. %A375 clearance and apoptosis are based on livecell imaging of A375 tumor cells and are calculated as described in the methods. IFNy was measured by MSD immunoassay.

Example 7

In Vitro Growth Inhibition Study by the Combination of HPK1 Inhibitor PF-07265028 and Anti-PD1 Antibody Nivolumab

The purpose of this example was to evaluate the potential of PF-07265028 with or without an anti-PD1 antibody of the present embodiments (nivolumab) as a treatment option for cancer.

This example was conducted according to the same protocol as described in Example 6 except to replace sasanlimab 2 pg/mL with nivolumab 10 pg/mL. Flow cytometry was conducted as described below.

Materials and Methods

Flow cytometry: For cytometry, T cells were washed from co-cultures at 60 hours, and stained for CD3, CD4, CD8, CD69, Ki67, and IFNy by antibody clones SK7 (BD 564001 ), SK3 (BD 612887), RPA-T8 (Biolegend 301028), FN50 (Biolegend 310932), B56 (BD 561284), and B27 (BD 554702) according to manufacturer’s protocols. Live cells were identified by Live/Dead Near-IR stain (ThermoFischer L10119). Stained cells imaged on a Cytek Aurora CS, and data analyzed on FlowJo (version 10).

Results:

FIG. 5 shows the effect of PF-07265028 alone and in combination with nivolumab on the growth of the A375 tumor cells. FIG. 6 shows the dose response of PF-07265028 alone and in combination with nivolumab on the clearance of the A375 tumor cells in a T cell co-culture. FIG. 7 shows the effect of PF-07265028 alone and in combination with nivolumab on T cell proliferation as measured by Ki67. FIG. 8 shows the effect of PF- 07265028 alone and in combination with nivolumab on IFNy production per activated T cell. PF-07265028 increases T cell proliferation in co-cultures with tumor cells while nivolumab increases the amount of IFNy produced per T cell. When PF-07265028 and nivolumab are combined, there is synergy through an increase in both T cell proliferation and IFNy production resulting in greater tumor growth control.

Example 8

In Vitro T Cell IFNy Production in Co-Culture with Breast Cancer Cells by the Combination of HPK1 Inhibitor PF-07265028 and Anti-PD1 Antibody Nivolumab

The purpose of this example was to evaluate the effects of the HPK1 inhibitor PF- 07265028 on the ability of tumor-reactive T cells to produce IFNy in response to co-culture with an additional tumor indication, MDA-MB-231 breast cancer tumor cells, and to evaluate the potential of PF-07265028 with or without an anti-PD1 antibody as a treatment option for cancer.

Materials and Methods

Cell Culture:

To generate tumor-reactive T cells, CD4 or CD8 T cells were isolated from PBMCs derived from two independent human donors, designated as ‘subject T and ‘subject 2’, following the EasySep Human T cell isolation kit (obtained from StemCell Technologies) protocol, and mixed with mitomycin treated MDA-MB-231 cells (10 pg/mL Mitomycin C for 2 hours; mitomycin C is obtained from StemCell Technologies) in X-VIVO™ 15 medium (obtained from Lonza Walkersville, Inc.) with 10% human serum AB (obtained from Fisher Scientific). After 10 minutes, 20 ng/mL recombinant human IL-2 (IS) and 10 ng/mL recombinant human IL-7 (obtained from Miltenyi Biotec) were added to expand activated T cells, and expansion was continued in G-Rex culture vessels for 21 days. The resulting cells were cryopreserved in solution of 90% fetal bovine serum (FBS, obtained from Fisher Scientific) and 10% DMSO.

MDA-MB-231 human breast cancer cells were passaged in Gibco Roswell Park Memorial Institute 1640 (RPMI) medium containing 10% FBS and 1 % penicillin/streptomycin. Prior to co-culture assays, the MDA-MB-231 cell line was transduced with Incucyte NucLight Green Lentivirus (nucGFP) (obtained from Sartorius) and selected by 1 pg/mL puromycin (obtained from Sigma).

Tumor Co-Culture assay Assay:

MDA-MB-231 cells expressing nucGFP were seeded at 5,000 to 10,000 cells per well in 100 pL RPMI with 10% FBS (assay medium) in an ImageLock 96-well plate (from Sartorius). Cells were cultured for 18 hours in a humidified incubator at 37°C and 5% CO2. The following day, frozen CD4 and CD8 cells were thawed in 37°C assay medium and rested for 2 hours. Meanwhile the media on the previously plated A375 cells was replaced with 50 pL assay medium containing 3x Annexin V-Red staining reagent and 3 mM CaCl2. 333 nM (3x final) concentration of PF-07265028 and/or 30 pg/mL (3x final) nivolumab (purchased from SelleckChemicals) was added to each well in 50 pL of assay medium, and T cells were added in 50 pL of assay medium as well. Human T cells from subject 1 and subject 2 were assayed independently. For subject 1 , equal amounts of CD4 and CD8 T cells were mixed, and then added to each well. For subject 2, only CD8 T cells were added to each well. In total, cells contained 10 T cells for every tumor cell, a 10:1 E:T ratio. Plates were then transferred to 37°C and 5% CO2 Incucyte S3 incubator and monitored for 4 days. Following 96 hours of co-culture, 50 pL of cell culture supernatant were collected from each well, transferred to a separate 96 well plate, and stored at -20°C overnight. The following day, supernatant samples are thawed at room temperature and analyzed by MSD quantitative immunoassay (Human IFNy Kit, Meso Scale Diagnostics) according to manufacturer’s protocol. Concentrations of IFNy in supernatants are quantitatively determined in units of pg/mL using manufacturer provided calibrators of defined concentrations to set a standard curve.

Results:

To assess the potential combination benefit of PF-07265028 with an anti-PD1 antibody, co-cultures of T cells and tumor cells were evaluated in the presence of nivolumab and PF-07265028. Tumor-reactive T cells from two human donors, subject 1 and subject 2, were generated against the MDA-MB-231 cell line and then co-cultured with MDA-MB-231 tumor cells as described above in the Materials and Methods. Cells were treated with 111 nM PF-07265028 alone, 10 pg/mL nivolumab alone, or the combination of those agents. The controls were either MDA-MB-231 cells cultured without T cells or cultured with mock-treated T cells (in DMSO). After 96 hours of coculture, supernatants were collected and analyzed by MSD quantitative immunoassay as described above in Materials and Methods. The effect of PF-07265028 alone and in combination with anti-PD1 antibody nivolumab on the amount of IFNy produced by subject 1 T cells in co-culture with MDA-MB-231 tumor cells is shown in FIG. 9. The control shows the amount of IFNy produced by subject 1 T cells in co-culture with MDA- MB-231 tumor cells in the presence of DMSO treatment (Mock) or DMSO with nivolumab. A ratio of ten T cells were plated for each tumor cell. Bar graphs depict the average of three replicates with error bars representing the SEM and individual data points plotted.

The effect of PF-07265028 alone and in combination with anti-PD1 antibody nivolumab on the amount of IFNy produced by subject 2 T cells in co-culture with MDA- MB-231 tumor cells is shown in FIG. 10. The control shows the amount of IFNy produced by subject 2 T cells in co-culture with MDA-MB-231 tumor cells in the presence of DMSO treatment (Mock) or DMSO with nivolumab. A ratio of ten T cells were plated for each tumor cell. Bar graphs depict the average of three replicates with error bars representing the SEM and individual data points plotted. Combination Benefit of PF-07265028 and Anti-PD1 Antibody Nivolumab in Enhancing IFNy Produced by T cells in Co-Culture with Breast Cancer Cells

It is observed that the addition of 111 nM PF-07265028 resulted in an increase in IFNy production, consistent with an increase in T cell activity. Addition of 111 nM PF- 07265028 to subject 1 T cells in co-culture with MDA-MB-231 tumor cells increased IFNy production from 9,114 pg/mL with mock-treated cells to 17,473 pg/mL (Table 3, FIG. 9). Comparatively, addition of 10 pg/mL anti-PD1 antibody nivolumab alone to subject 1 T cells in co-culture with MDA-MB-231 tumor cells increased IFNy production from 9,114 pg/mL with mock-treated cells to 16,012 pg/mL (Table 3, FIG. 9). Combined addition of 111 nM PF-07265028 and 10 pg/mL nivolumab further increased IFNy production to 29,795 pg/mL (Table 3, FIG. 9). Addition of 111 nM PF-07265028 to subject 2 T cells in co-culture with MDA-MB-231 tumor cells increased IFNy production from 231 pg/mL with mock-treated cells to 816 pg/mL (Table 3, FIG. 10). Comparatively, addition of 10 pg/mL anti-PD1 antibody nivolumab alone to subject 2 T cells in co-culture with MDA-MB-231 tumor cells increased IFNy production from 231 pg/mL with mock-treated cells to 398 pg/mL (Table 3, FIG. 10). Combined addition of 111 nM PF-07265028 and 10 pg/mL nivolumab further increased IFNy production to 1 ,106 pg/mL (Table 3, FIG. 10). All quantified results for both human donors are tabulated in Table 3 and graphed in FIGs. 9 and 10 and demonstrate a combination benefit of PF-07265028 and anti-PD1 antibody nivolumab in enhancing IFNy production by T cells in co-culture with breast cancer tumor cells.

Table 3: Combination Benefit of PF-07265028 and anti-PD1 at 96 Hours

Tabulated above are values plotted in FIGs. 9 and 10, and represent the average and SEM of three replicates. IFNy was measured by MSD immunoassay as described in Methods.

All references cited herein, including patent applications, patent publications cited in the specification are herein incorporated by reference in their entirety. Although the foregoing disclosure has been described in some detail by way of illustration and example, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The foregoing description and Examples detail certain specific embodiments of the disclosure and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the disclosure may be practiced in many ways and the disclosure should be construed in accordance with the appended claims and any equivalents thereof.