PD-1 variant immunomodulatory proteins and uses thereof

1. A variant PD-1 polypeptide comprising an IgV domain, wherein:

(a) the variant PD-1 polypeptide comprises one or more amino acid substitutions in an unmodified PD-1 polypeptide selected from the amino acid substitutions P8T, D9E, D9G, D9N, D9V, P11A, W12G, W12L, W12R, N13Y, P14H, P14L, P14S, T16A, T16I, T16S, F17I, F17V, F17Y, S18T, A20S, A20T, A20V, L21V, L22I, V23E, V23G, V24L, D28E, N29D, T31I, T31N, T31S, T33I, C34Y, S35N, F36I, F36L, F36Y, S37P, S37T, N38S, N38T, T39R, T39S, S40P, S40T, E41D, E41V, S42G, S42R, F43L, F43Y, V44M, Y48N, M50T, S51G, P52A, P52L, S53T, N54H, Q55R, T56M, T56P, T56S, K58R, L59M, L59R, L59V, E64D, E64K, R66H, R66S, S67C, S67I, S67N, S67R, P69H, G70C, G70E, G70S, Q71H, Q71K, Q71L, D72N, C73A, C73G, C73H, C73P, C73Y, F75Y, R76H, R76S, V77D, T78S, Q79P, L80Q, P81S, R84H, R84Q, D85G, D85N, F86Y, H87L, S89G, S89N, V90L, V90M, V91A, V91D, V91I, R92N, R92S, A93V, R94Q, R95L, N96T, T100A, T100I, T100S, L102F, G104A, G104T, G104V, A105C, A105G, A105L, I106L, L108T, A109G, K111M, K111N, Q113R, Q113W, I114T, K115D, K115E, K115I, K115N, K115Q, E116D, R119H, R119L, R119P, R119Q, R119W, T125K, T125S, R127S, R128M, A129S, E130K, V131A, V131E, P132H, P132R, P132S, P132T, T133A, T133R, T133S, A134D, A134V, H135N, H135R, H135Y, P136L, P136T, S137C, P138S, P138T, S139T, P140A, P140L, P140R, R141G, R141M, R141S, R141W, P142A, P142L, P142R, P142T, A143D, A143S, A143V, G144D, or G144S with reference to numbering of positions set forth in SEQ ID NO:37; or

(b) the variant PD-1 polypeptide comprises two or more amino acid substitutions in an unmodified PD-1 polypeptide selected from C73A, C73G, C73H, C73P, C73R, C73S, C73Y, F86Y, S107T, A112V, K115D, K115E, K115I, K115N, K115Q, R119H, R119L, R119P, R119Q, R119W or A120V, with reference to numbering of positions set forth in SEQ ID NO:37;

wherein the unmodified PD-1 polypeptide comprises (i) the sequence of amino acids set forth in any of SEQ ID NOS: 37, 244, or 392, (ii) a sequence of amino acids that has at least 95% sequence identity to any of SEQ ID NOS:37, 244, or 392; or (iii) is a portion of (i) or (ii) comprising an IgV domain.

2. The variant PD-1 polypeptide of claim 1 , wherein the one or more amino acid substitutions or the two or more amino acid substitutions are selected from A112V/R119L/A120V; N13S/A120V/P142A; C34Y/N54D/T100S/A112V/A120V; M50V/S67N/L80Q/A120V/A143S; A112V/R119W; R84H/H87L/A112V/R119W; Q71R/V91I/A112V/R119L/A120V/T125S; A112V/K115E; M50L/L59V/R66H/A112V/H135Y/P138T/P142L; R119W; S53G/Q55R/A112V/K115E/A120V/S139T; R119W/H135R; A120V/T125I; A112V/A120V/V131A; F17I/K111E/A112V/A120V; S18T/R119Q/R141M; F36L/S37T/A112V/H135N/P138S; A112V/T125I; M50I/A112V/A120V; S67N/C73R/A93V/A112V/A120V; D72G/A112V/A120V; N96D/A112V/A120V/T125S; F86Y/R119W/T125I; R119P/T133R; K111M/A112V/K115E/P132H; S67G/A112V/T125I/T133S; A112V/A120V; S37P/A112V/R119W; W12L/S37P/A112V/R119W; D9G/A112V/A120V; T31S/S37T/A112V/T125I/A143S; S37T/A112V/T1251; R92G/A112V/A120V; E64D/F86Y/A112V/A120V; H87R/A112V/R119W; N13D/A105V/A112V/A120V/A134D; A112V/R119L/A120V/S137C; T16I/M50I/A112V/A120V; M50L/A112V/R119Q/A120V/T125I/H135R; D57V/A112V; S67N/R119W; S67N/A112V/A120V; N54Y/A112V/P140A; F43Y/P69L/R119W; N54Y/A112V/R119W; T56M/C73S/R76H/A112V/R119L/A120V/P132T/R141W; F17I/S40P/E41D/S67N/R95L/A112V/A120V/T125I/R141M; F17L/T31S/S35N/P81S/N96S/A112V/R119W; F43L/S67N/C73R/A112V/A120V; W12L/N38D/A112V/R119Q/A120V/P142T; S67N/P69H/C73R/Q79P/V91D/A112V/A120V/P136T/A143D; F17I/S40P/S67N/Q79R/A112V/R119W/T125I; F43Y/M50V/S67N/C73R/R92G/A112V/A120V/P136T; F17L/T56M/S67N/A112V/R119W/A120V/P142R; W12L/N54Y/S67N/F75Y/V91D/R95L/G104A/A112V/R119W/R141M; F17L/S37T/S67N/T78S/F86Y/A112V/R119H/A120V/V131E/A143V; N13D/S40P/A112V/R119L/A120V/S137C; F17V/A30V/E41V/R76S/A112V/R119Q/A120V/V131A; F17I/T25A/M50V/S53T/R66S/S67R/S107T/A112V/R119W/A143V; N13D/S40P/S67N/C73R/R95L/G104A/A112V/A120V; S40P/T56A/S67N/C73R/A112V/R119Q/A120V/V131A; N13Y/S40P/F43L/Q68P/R92G/A112V/R119L/A120V; F17L/S67N/Q71L/C73S/A112V/R119Q/A120V/P142L; F17I/S40P/P69S/C73S/N96S/G104A/A112V/A120V; F17I/S40P/A112V/R119L/A120V/P140R; A112V/A120V/T133S; A20S/S67N/C73R/R94Q/A112V/R119Q/A120V/T125I/P132S; N13D/S67N/C73R/R95L/A112V/R119Q/A120V/T125I; S40P/S67N/C73R/N96T/A112V/A120V; L21V/S40P/R95L/G104A/A112V/A120V/A129S/V131A/R141G; P14S/S40P/S42R/P52A/T56M/A112V/R119W/T125I/P142A; S40P/F43L/T56A/S67N/C73S/A112V/R119L/A120V; F17I/S40P/M50V/S67N/C73S/R95L/G104A/A112V/R119L/A120V; S40P/T56M/C73S/R95L/A112V/R119W/T125I/V131A/R141W; F171/A20V/S51G/N54D/F86Y/A112V/A120V/T125I; F17I/T31N/T56M/S67N/C73R/G104A/A112V/R119Q/A120V/T133A/P140L; F17V/S40P/R92G/R95L/A112V/R119W; W12G/F17L/T56V/S67N/A112V/R119W/V131E/R141S; F86Y/R92G/A112V/R119L/A120V/T125K/T133S; P8T/F17I/S67N/F86Y/G104A/A112V/A120V/S139T; F17I/S40P/A112V/R119W/G144D L22I/S67I/G70S/Q71R/S107T/A112V/R119L/A120V/T125I/T133S; S40P/A112V/A120V; T16S/S67N/C73R/A112V/R119W; N13D/S40P/A112V/A120V/T125I/T133S; M50L/V91D/A112V/R119W/P132R/R141G; F17I/S40P/M50I/S67R/A112V/R119L/A120V/S137C; S40P/A112V/R119W; F17I/V24L/A112V/R119W; S40P/S67N/C73R/A93V/A112V/A120V; N13D/F17I/A112V/R119W/R141M; S67N/C73R/A112V/A120V/R141S; W12R/L59V/R66H/F86Y/V90L/A112V/K115N/R119L/A120V; F36L/M50I/S51G/C73R/S107T/K111M/A120V/V131E; M50V/R119W/A120V/T125I/R141G; A109G/A112V/K115E/R119W; Q55R/R76H/A112V/K115I/A120V; W12R/F86Y/R95L/A112V/R119P/T133R; S67N/C73Y/A112V; F17I/S40P/T56S/A112V; W12L/P14S/M50V/S67R/A93V/R94Q/K111T/A112V/R119W/A120V; F36L/L59R/S67N/A105L/A112V/R119W/A120V/A143V; L59M/E64K/F86Y/R94Q/A112V/R119L/A120V/T125I/T133S; P14H/F17Y/T39R/S40T/K58R/V77D/G104V/A112V/Q113R/1114T/A143V; D57V/L59M/P69S/C73S/A112V/R119L/A120V/A134V; V23E/T39S/S40P/C73Y/V91A/R92N/L102F/A112V/R119W/A120V; T16A/F17I/F36L/S67N/C73R/H87L/R92G/R95L/A112V/R119W; C73S/A105V/A112V/R119W/A120V/T133A; P14S/M50L/L59V/R66H/G70S/C73R/A112V/A120V/T125I/G144S; P8T/S40P/S53N/R95L/A112V/A120V/T125I/R128M/P138T/R141G; S37T/T56M/C73S/D85N/A109G/A112V/A120V/H135Y/A143S; S67N/C73R/A93V/G104A/A112V/R119Q/R127S/H135R; S40P/S67N/C73R/A112V/A120V; S67N/C73R/A93V/A112V/R119W; C73R/A93V/A112V/R119W; S67N/C73R/A112V/A120V; N13D/P69H/C73R/A93V/A112V/R119W/R141S; F17I/N38D/S40P/S67N/C73R/A112V/A120V; N13D/F17I/S40P/T56M/R66S/S67N/G70C/A112V/A120V/R141S; S40P/F43L/R66S/S67N/G70C/A112V/R119L/A120V; V23G/S40T/G104A/A112V/R119L/A120V; W12R/F36I/N54Y/S67I/C73S/A93V/G104A/A112V/K115Q/R119W; S40P/M50L/S67I/C73S/R92G/A93V/A112V/A120V/R141S; F17I/T25A/S40P/F43Y/S67N/C73R/F86Y/A112V/R119W/T125I; F17I/S40P/M50L/S67I/C73S/A112V/A120V/E130K/P136L; W12R/S40P/S67N/C73R/A93V/A112V/A120V/R141S; F17I/S40P/M50L/S67I/C73S/R92G/A93V/A112V/A120V/R141S; F17I/S40P/M50L/S67I/A112V/A120V/R141S; V23G/T56P/S67I/C73S/F86Y/R92G/G104A/A112V/R119W/A143D; F17I/S40P/M50L/S67I/C73S/R92G/A93V/A112V/R119W/T125I; D9V/P11A/N13D/S40T/T56M/S67N/R95L/G104A/A112V/R119L/A120V; F17I/T25A/S40P/F43Y/S67N/A112V/R119L/A120V; D9E/F17I/S40P/M50L/S67I/C73S/V90M/T100A/G104A/K111M/A112V/R119W/A120V/A143D; F17I/S40P/M50L/S67I/C73S/R92G/A93V/A112V/A120V/R141G; F17I/S40P/S67N/S89G/R95L/A112V/R119W/T125I; F17I/S40P/M50L/S67I/C73S/R92G/A93V/K111M/A112V/R119Q/A120V; D9N/F17I/T31N/S40P/M50L/S67I/C73S/R92G/A93V/A112V/A120V/R141S; N13D/S40T/T56M/S67N/R95L/G104A/K111R/A112V/A120V/T125I; W12G/F17I/S40P/M50L/A112V/P132T; D9N/F17I/S40P/M50T/T56A/G70S/C73R/R92G/A93V/A112V/A120V/R141S; N38S/L59M/Q71L/S107T/A112V/K115E/R119Q/A120V; M50V/L59M/A112V/R119Q/A120V P81S/F86Y/R92S/S107T/A112V/K115E/A120V; C73R/R84Q/F86Y/S107T/A112V/K115D/A120V; W12G/M50T/S53N/S67R/G70S/C73R/G104V/A112V/K115E/R119W; T31I/F36Y/F43Y/S67N/G70S/C73R/V90M/A112V/R119W; R76S/S107T/K111N/A112V/K115N/A120V; F86Y/A112V/R119W/A120V; N13S/M50I/R76S/S107T/A112V/K115N/R119W/A120V; P14H/T16S/M50L/C73R/R84Q/F86Y/S107T/A112V/K115E/A120V; F17L/T25A/L59M/E64K/F86Y/R94Q/S107T/A112V/K115N/R119W/A120V; Q71R/F86Y/A112V/K115E/R119Q/A120V; R76S/S107T/A112V/K115N/R119W/A120V; M50I/A112V/K115D/A120V; M50V/P81S/F86Y/R92S/S107T/A112V/K115E/A120V; M50I/S67N/G70R/K111T/A112V/R119W/A120V; P14H/T16S/M50L/L80Q/K111M/R119Q/A120V; T31I/F36Y/E64K/A112V/K115E/R119Q/A120V; S107T/A112V/R119W/A120V; T56S/A112V/K115E/A120V; T56S/A112V/R119W; N46I/Y48N/D57Y/S67C/V90L/A112V; T56S/A112V/R119P; P14H/F17I/V44M/A112V/K115E/A120V; N38S/T56S/A112V/K115E/A120V; S42G/M50L/P69S/F86Y/A112V/K115E; P14H/T56S/A112V/K115E/A120V; N13S/S67N/G70C/F86Y/S89N/V91D/A112V/R119L/A120V; W12G/S67N/Q71R/F86Y/K111M/A112V/K115Q/R119W; S67N/C73R/V91D/S107T/K111M/A112V/K115Q/R119W; N13S/M50I/R76S/S107T/K111M/A112V/K115Q/R119W; T33I/S67N/G70S/S107T/K111N/A112V/K115E/R119W; P69L/F86Y/V90M/T100I/S107T/K111N/A112V/K115N/A120V; F17L/T25A/P69L/F86Y/V90M/T100I/S107T/K111N/A112V/K115N/A120V; T33I/M50I/R76S/F86Y/S107T/A112V/K115N/R119W/A120V; N13S/S67N/C73R/F86Y/S107T/A112V/Q113R/K115E/A120V; S67N/C73R/F86Y/V91D/S107T/A112V/K115D/A120V; F17L/T25A/S67N/C73R/R84Q/F86Y/A93V/A112V/K115E/R119W; T56S/A112V/K115E P69L/V91D/A112V/K115N/R119W/A120V; N13S/E41D/M50I/G70V/D72N/F86Y/R94Q/A112V/R119L/A120V; M50I/P69L/F86Y/V90M/T100I/S107T/K111N/A112V/K115N/A120V; M50I/C73R/S107T/K111N/A112V/K115N/A120V; F86Y/A112V/K115N/R119W; M50I/S67N/C73R/F86Y/R95L/S107T/A112V/K115N/R119W; M50L/Q68R/P69S/F86Y/S107T/A112V/K115N/R119W; N38T/A112V/K115N/R119L/A120V; S67N/Q71H/F86Y/R95L/S107T/A112V/K115N/R119W; A20T/D28E/F36L/M50I/Q68R/P69S/F86Y/A112V/R119L/A120V; M50I/S67N/C73R/F86Y/R95L/A112V/Q113W/R119L/A120V; L59M/S67N/Q71L/C73R/R95L/S107T/A112V/K115N/R119W; P52L/S53N/C73S/A112V/E116D/R119W; Q71R/C73R/A112V/K115N/R119L/A120V; W12L/A20T/N29D/S37P/L59M/Q68R/P69S/F86Y/A112V/R119L/A120V; N46I/Y48F/D57V/P69L/A112V/K115N/R119W; M50I/S67N/C73R/F86Y/R95L/S107T/A112V/K115Q/R119Q/A120V; N54D/P69H/C73R/F86Y/R95L/S107T/A112V/K115N/R119W; T56S/Q71K/F86Y/R95L/S107T/A112V/K115N/R119W; C73A/F86Y/S107T/A112V/K115N/R119Q/A120V; C73R/F86Y/A105G/S107T/A112V/K115N/R119L/A120V; N54H/G70E/C73P/F86Y/A112V/K115D/R119L/A120V; C73G/F86Y/S107T/A112V/K115N/R119Q/A120V; N54S/C73G/F86Y/S107T/A112V/K115D/R119L/A120V; F86Y/S107T/A112V/K115D/R119W/A120V; G70E/C73P/F86Y/S107T/A112V/K115E/R119Q/A120V; C73G/F86Y/A105G/S107T/A112V/K115D/R119W; C73G/F86Y/S107T/A112V/K115D/R119L/A120V; C73S/F86Y/S107T/A112V/K115D/R119L/A120V; L45V/C73G/F86Y/G104A/S107T/A112V/K115N/R119W/A120V; C73P/F86Y/S107T/A112V/K115D/R119Q/A120V; C73S/F86Y/S107T/A112V/K115E/R119Q/A120V; C73S/F86Y/G104T/S107T/A112V/K115E/R119W/A120V; C73R/F86Y/S107T/K111R/A112V/K115D/A120V; P14L/C73G/F86Y/S107T/A112V/K115D/R119L/A120V; G70E/F86Y/S107T/A112V/K115D/R119L/A120V; C73G/F86Y/G104V/S107T/A112V/K115N/R119L/A120V; C73S/F86Y/G104S/S107T/L108F/A112V/K115D/R119L/A120V; C73S/F86Y/S107T/A112V/K115D/A120V; C73R/F86Y/S107T/A112V/K115D/R119L/A120V; C73S/F86Y/S107T/A112V/Q113R/K115D/R119L/A120V; C73S/F86Y/V91A/S107T/A112V/K115D/R119L/A120V; G70E/C73P/F86Y/A105G/S107T/A112V/K115D/R119Q/A120V; C73G/F86Y/A105G/S107T/A112V/K115D/R119L/A120V; C73G/F86Y/A105G/S107T/A112V/Q113R/K115D/R119L/A120V; F86Y/S107T/A112V/K115D/R119Q/A120V; C73R/F86Y/S107T/A112V/K115N/R119L/A120V; C73A/F86Y/S107T/A112V/Q113R/K115E/R119Q/A120V; C73R/F86Y/S107T/A112V/K115D/R119Q/A120V; C73G/F86Y/A112V/K115D/R119W/A120V; C73P/F86Y/A105G/S107T/A112V/Q113R/K115D/R119L/A120V; C73R/F86Y/A105G/S107T/A112V/K115D/R119L/A120V; C73A/F86Y/S107T/A112V/K115D/R119L/A120V; P69S/C73R/F86Y/S107T/A112V/K115D/R119W/A120V; C73S/F86Y/G104S/S107T/A112V/K115E/R119W/A120V; Q68R/C73S/F86Y/S107T/A112V/K115D/R119Q/A120V; C73R/F86Y/S107T/A112V/K115N/R119Q/A120V; G70E/C73R/F86Y/S107T/A112V/K115N/R119Q/A120V; C73S/F86Y/S107T/A112V/K115D/R119W/A120V; G70E/F86Y/G104T/I106L/S107T/L108T/A112V/K115N/R119L/A120V; C73H/F86Y/A105G/S107T/A112V/K115D/R119L/A120V; G70E/C73P/F86Y/A105C/S107T/A112V/K115D/R119L/A120V; G70E/C73P/F86Y/S107T/A112V/K115D/R119Q/A120V; C73S/F86Y/S107T/K111R/A112V/K115E/R119L/A120V; C73R/D85G/F86Y/A105G/S107T/A112V/K115D/R119Q/A120V; C73R/F86Y/S107T/A112V/K115E/R119W/A120V; N54S/C73G/F86Y/S107T/A112V/K115E/R119Q/A120V; C73S/F86Y/G104S/S107T/A112V/K115N/R119L/A120V; F17L/Q71R/C73G/F86Y/A105G/S107T/A112V/K115D/R119L/A120V; C73G/F86Y/S107T/A112V/K115E/R119W/A120V; G70E/C73G/F86Y/A105C/S107T/A112V/K115E/R119L/A120V; C73G/F86Y/G104A/S107T/A112V/K115D/R119W/A120V; C73S/F86Y/S107T/A112V/K115N/A120V; C73P/F86Y/S107T/A112V/K115N/R119L/A120V; W12R/F86Y/S107T/A112V/K115D/R119Q/A120V; G70E/C73G/F86Y/A105L/A112V/K115N/R119Q/A120V; or F86Y/S107T/A112V/K115N/R119W/A120V, with reference to numbering of positions set forth in SEQ ID NO:37.

3. The variant PD-1 polypeptide of claim 1 , wherein the variant PD-1 polypeptide is or comprises the IgV domain.

4. The variant PD-1 polypeptide of claim 1 , wherein the variant PD-1 exhibits increased binding to the ectodomain of human PD-L1 compared to the binding affinity of the unmodified PD-1 for the ectodomain of human PD-L1.

5. An immunomodulatory protein comprising a variant PD-1 polypeptide of claim 1 linked to a multimerization domain.

6. The immunomodulatory protein of claim 5 , wherein the multimerization domain is an Fc domain or is a variant Fc domain with reduced effector function.

7. A conjugate, comprising a variant PD-1 polypeptide of claim 1 linked to a targeting moiety that specifically binds to a molecule on the surface of a cell.

8. An engineered cell, comprising a variant PD-1 polypeptide of claim 1 ; an immunomodulatory protein comprising a variant PD-1 polypeptide of claim 1 , or a conjugate comprising a variant PD-1 polypeptide of claim 1 .

9. An infectious agent, comprising a variant PD-1 polypeptide of claim 1 , an immunomodulatory protein comprising a variant PD-1 polypeptide of claim 1 , or a conjugate comprising a variant PD-1 polypeptide of claim 1 .

10. A pharmaceutical composition, comprising a variant PD-1 polypeptide of claim 1 , an immunomodulatory protein comprising a variant PD-1 polypeptide of claim 1 , a conjugate comprising a variant PD-1 polypeptide of claim 1 , or an engineered cell comprising a transmembrane protein comprising a variant PD-1 polypeptide of claim 1 .

11. An article of manufacture comprising the pharmaceutical composition of claim 10 in a vial or a container.