Novel proteins specific for pyoverdine and pyochelin

Abstract

The present disclosure provides hNGAL muteins that bind a pyoverdine family member or pyochelin and can be used in various application including pharmaceutical applications, for example, to inhibit or reduce growth of P. aeruginosa . The present disclosure also concerns methods of making one or more pyoverdine- or pyochelin-binding muteins described herein as well as compositions comprising one or more of such muteins. The present disclosure further relates to nucleic acid molecules encoding such muteins and to methods for generation of such muteins and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of these muteins as well as compositions comprising one or more of such muteins.

1 - 12 . (canceled) 
     
     
         13 . A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a human neutrophil gelatinase-associated lipocalin (hNGAL) mutein polypeptide having binding specificity for pyoverdine type III, wherein the hNGAL mutein comprises 10 or more mutations at positions 36, 40, 41, 42, 49, 52, 65, 68, 70, 73, 77, 79, 81, 87, 96, 100, 103, 105, 106, 127, and 134 of SEQ ID NO: 1. 
     
     
         14 . A host cell containing a nucleic acid molecule of  claim 13 . 
     
     
         15 . The nucleic acid molecule of  claim 13 , wherein the nucleic acid molecule encodes an hNGAL mutein comprises ten or more of the following mutations of SEQ ID NO: 1: L36E, A40L, I41A, L42R, Q49M, Y52M, N65D, S68E, L70R, K73H, D77K, W79D, R81V, C87S, N96L, Y100D, L103Q, S105P, Y106E, S127V, K134W. 
     
     
         16 . The nucleic acid molecule of  claim 13 , wherein the nucleic acid molecule encodes an hNGAL mutein comprises SEQ ID NO: 53 
     
     
         17 . The nucleic acid molecule of  claim 13 , wherein the nucleic acid molecule encodes an hNGAL mutein that has binding specificity for pyoverdine type III succinyl, pyoverdine type III succinamid, and/or pyoverdine type III α-ketoglutaryl. 
     
     
         18 . The nucleic acid molecule of  claim 13 , wherein the nucleic acid molecule encodes an hNGAL mutein that is capable of binding pyoverdine type III complexed with iron with a K D  of about 200 nM or lower. 
     
     
         19 . The nucleic acid molecule of  claim 13 , wherein the nucleic acid molecule encodes an hNGAL mutein comprises 12 or more mutations at positions 36, 40, 41, 42, 49, 52, 65, 68, 70, 73, 77, 79, 81, 87, 96, 100, 103, 105, 106, 127, and 134 of SEQ ID NO: 1. 
     
     
         20 . A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a human neutrophil gelatinase-associated lipocalin (hNGAL) mutein polypeptide having binding specificity for pyoverdine type III, wherein the hNGAL mutein comprises 15 or more mutations at positions 36, 40, 41, 42, 49, 52, 65, 68, 70, 73, 77, 79, 81, 87, 96, 100, 103, 105, 106, 127, and 134 of SEQ ID NO: 1. 
     
     
         21 . A host cell containing a nucleic acid molecule of  claim 20 . 
     
     
         22 . The nucleic acid molecule of  claim 20 , wherein the nucleic acid molecule encodes an hNGAL mutein comprises 15 or more of the following mutations of SEQ ID NO: 1: L36E, A40L, I41A, L42R, Q49M, Y52M, N65D, S68E, L70R, K73H, D77K, W79D, R81V, C87S, N96L, Y100D, L103Q, S105P, Y106E, S127V, K134W. 
     
     
         23 . The nucleic acid molecule of  claim 20 , wherein the nucleic acid molecule encodes an hNGAL mutein comprises SEQ ID NO: 53. 
     
     
         24 . The nucleic acid molecule of  claim 20 , wherein the nucleic acid molecule encodes an hNGAL mutein that has binding specificity for pyoverdine type III succinyl, pyoverdine type III succinamid, and/or pyoverdine type III α-ketoglutaryl. 
     
     
         25 . The nucleic acid molecule of  claim 20 , wherein the nucleic acid molecule encodes an hNGAL mutein that is capable of binding pyoverdine type III complexed with iron with a K D  of about 200 nM or lower. 
     
     
         26 . A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a human neutrophil gelatinase-associated lipocalin (hNGAL) mutein polypeptide having binding specificity for pyoverdine type III, wherein the hNGAL mutein comprises SEQ ID NO: 53. 
     
     
         27 . The nucleic acid molecule of  claim 26 , wherein the nucleic acid molecule encodes an hNGAL mutein consists of SEQ ID NO: 53. 
     
     
         28 . A host cell containing a nucleic acid molecule of  claim 26 . 
     
     
         29 . The nucleic acid molecule of  claim 26 , wherein the nucleic acid molecule encodes an hNGAL mutein that has binding specificity for pyoverdine type III succinyl, pyoverdine type III succinamid, and/or pyoverdine type III α-ketoglutaryl. 
     
     
         30 . The nucleic acid molecule of  claim 26 , wherein the nucleic acid molecule encodes an hNGAL mutein that is capable of binding pyoverdine type III complexed with iron with a K D  of about 200 nM or lower.