Markers of amyotrophic lateral sclerosis survival and uses thereof

Abstract

Provided herein are markers and uses thereof for predicting survival in amyotrophic lateral sclerosis (ALS) patients, determining impact of an ALS therapy on survival of an ALS patient, and predicting the rate of ALS progression.

Description

Atty. Docket No.14678-018-228 MARKERS OF AMYOTROPHIC LATERAL SCLEROSIS SURVIVAL AND USES THEREOF 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 535,524, filed August 30, 2023, which is incorporated by reference herein in its entirety. 2. SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14678-018-228_SEQLISTING.xml”, was created on August 20, 2024, and is 5,086 bytes in size. 3. FIELD

[0003] Provided herein are markers and uses thereof for predicting survival in amyotrophic lateral sclerosis (ALS) patients, determining impact of an ALS therapy on survival of an ALS patient, and predicting the rate of ALS progression. 4. BACKGROUND

[0004] Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease resulting from neurodegeneration and leads to progressive loss of motor neurons. ALS is characterized by heterogeneous clinical presentations and rates of progression among affected individuals. ALS progression and survival time can vary greatly between ALS patients. Currently relied upon methods of predicting an individual patient’s survival from diagnosis rely on subjective tools. As such, an objective tool to predict survival in ALS is needed in the field. 5. SUMMARY

[0005] In one aspect, the present disclosure provides a method of predicting survival in a subject having or suspected of having amyotrophic lateral sclerosis (ALS), comprising: (a) determining a level of 4-hydroxynonenal (4-HNE) in a plasma or serum sample from the subject; and (b) diagnosing the subject as: (i) a long-term ALS survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short-term ALS survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE.

[0006] In another aspect, the present disclosure provides a method of predicting survival in a subject having or suspected of having amyotrophic lateral sclerosis (ALS), comprising: (a) NAI-1540949621v1 1Atty. Docket No.14678-018-228 obtaining or having obtained a plasma or serum sample from the subject; (b) determining a level of 4-hydroxynonenal (4-HNE) in the subject; and (c) diagnosing the subject as: (i) a long-term survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4- HNE; or (ii) a short-term survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE.

[0007] In certain embodiments, the methods of predicting survival in a subject having or suspected of having ALS further comprise: (d) administering: (i) a first therapeutic regimen to the subject if the subject is a long-term survivor, or (ii) a second therapeutic regimen to the subject if the subject is a short-term survivor.

[0008] In certain embodiments, determining the level of 4-HNE comprises an assay that uses a composition comprising a solid surface having an antibody or an antigen-binding portion thereof that binds to a 4-HNE epitope.

[0009] In certain embodiments, the level of the 4-HNE in the sample equal to or lower than a reference range for 4-HNE correlates to an increased mean survival time for the subject. In certain embodiments, the level of the 4-HNE in the sample greater than the reference range for 4- HNE correlates to a decreased mean survival time for the subject.

[0010] In certain embodiments, the level of the 4-HNE in (c)(i) correlates with an ALSFRS- R rate of decline less than about one point per month. In certain embodiments, the level of the 4- HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about 0.7 points per month.

[0011] In certain embodiments, the level of the 4-HNE in (c)(ii) correlates with an ALSFRS- R rate of decline greater than about one point per month. In certain embodiments, the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about 1.2 points per month.

[0012] In certain embodiments, the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL.

[0013] In certain embodiments, the first therapeutic regimen and the second therapeutic regimen comprise an ALS therapy. In certain embodiments, the first therapeutic regimen and the second therapeutic regimen comprise the same ALS therapy. In certain embodiments, the first therapeutic regimen and the second therapeutic regimen comprise different ALS therapies.

[0014] In certain embodiments, the second therapeutic regimen comprises a palliative care therapeutic regimen. NAI-1540949621v1 2Atty. Docket No.14678-018-228

[0015] In certain embodiments, the second therapeutic regimen comprises a higher dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen. In certain embodiments, the second therapeutic regimen comprises a lower dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

[0016] In certain embodiments, the second therapeutic regimen comprises administering a dosage of the ALS therapy with increased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen. In certain embodiments, the second therapeutic regimen comprises administering a dosage of the ALS therapy with decreased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

[0017] In certain embodiments, the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol (e.g., Relyvrio, AMX0035), edaravone, riluzole, interleukin-2 (IL- 2), abatacept (CTLA-4 Ig), or any combination thereof. In certain embodiments, the IL-2 is an IL-2 conjugate or an IL-2 mutein. In certain embodiments, the ALS therapy comprises an IL- 2 / abatacept combination therapy. In certain embodiments, the ALS therapy comprises a low dose of IL-2. In certain embodiments, the low dose of IL-2 comprises a dose of IL-2 less than about 5.0 ×106IU / m2 / day. In certain embodiments, the IL-2 is a human recombinant IL 2. In certain embodiments, the IL-2 is aldesleukin.

[0018] In certain embodiments, the first ALS therapy or the second ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol (e.g., Relyvrio, AMX0035), edaravone, riluzole, IL-2, abatacept (CTLA-4 Ig), or any combination thereof.

[0019] In certain embodiments, the palliative care comprises management of one or more symptoms of ALS.

[0020] In certain embodiments, the sample was obtained from the subject at ALS diagnosis.

[0021] In another aspect, the present disclosure provides a method of determining impact of an ALS therapy on survival of a subject having or suspected of having ALS, comprising: (a) determining a level of 4-hydroxynonenal (4-HNE) in a plasma or serum sample from the subject; (b) diagnosing the subject as: (i) a long-term ALS survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short-term ALS survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE; (c) administering an ALS therapy to the subject; (d) determining a level of 4-HNE in a plasma or NAI-1540949621v1 3Atty. Docket No.14678-018-228 serum sample from the subject following (c); and (e) comparing the level of 4-HNE in (a) to the 4-HNE level in (d), thereby determining the impact of the ALS therapy on survival of the subject.

[0022] In certain embodiments, the determining the level of 4-HNE comprises an assay that uses a composition comprising a solid surface having an antibody or an antigen-binding portion thereof that binds to a 4-HNE epitope. In certain embodiments, the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL. In certain embodiments, the reference range for 4-HNE is about 0.5 µg / mL to about 15 µg / mL.

[0023] In certain embodiments, the level of the 4-HNE in the sample equal to or lower than a reference range for 4-HNE correlates to an increased mean survival time for the subject.

[0024] In certain embodiments, the level of the 4-HNE in the sample greater than the reference range for 4-HNE correlates to a decreased mean survival time for the subject.

[0025] In certain embodiments, the ALS therapy is predicted to improve survival of the subject when the 4-HNE level in (d) is equal to or less than the level of 4-HNE in (a).

[0026] In certain embodiments, the ALS therapy is predicted to have no effect or worsen survival of the subject when the 4-HNE level in (d) is higher than the level of 4-HNE in (a).

[0027] In certain embodiments, the method of determining impact of an ALS therapy on survival of a subject having or suspected of having ALS further comprises: (f) administering: (i) a continued dosage of the ALS therapy administered in (c) to the subject if the ALS therapy improves survival of the subject, or (ii) a second therapeutic regimen to the subject if the ALS therapy has no effect or worsens survival of the subject.

[0028] In certain embodiments, the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol (e.g., Relyvrio, AMX0035), edaravone, riluzole, interleukin-2 (IL- 2), abatacept (CTLA-4 Ig), or any combination thereof. In certain embodiments, the IL-2 is an IL-2 conjugate or an IL-2 mutein. In certain embodiments, the ALS therapy comprises an IL- 2 / abatacept combination therapy. In certain embodiments, the ALS therapy comprises a low dose of IL-2. In certain embodiments, the low dose of IL-2 comprises a dose of IL-2 less than about 5.0 ×106IU / m2 / day. In certain embodiments, the IL-2 is a human recombinant IL 2. In certain embodiments, the IL-2 is aldesleukin.

[0029] In certain embodiments, the second therapeutic regimen comprises a palliative care therapeutic regimen. In certain embodiments, palliative care comprises management of one or NAI-1540949621v1 4Atty. Docket No.14678-018-228 more symptoms of ALS.

[0030] In certain embodiments, the subject is naïve to ALS therapy.

[0031] In certain embodiments, the subject has previously received at least one ALS therapy.

[0032] In another aspect, the present disclosure provides a method of predicting the rate of ALS progression to disease endpoints in a subject having or suspected of having ALS, comprising: (a) obtaining or having obtained a plasma or serum sample from the subject; (b) determining a level of 4-hydroxynonenal (4-HNE) in a plasma or serum sample from the subject; and (c) diagnosing the subject as: (i) a slow ALS progressor if the level of the 4-HNE in the sample is equal to or lower than a reference range; or (ii) a rapid ALS progressor if the level of the 4-HNE in the sample is greater than the reference range, wherein the disease endpoints are death or a need for implementation of invasive respiratory measures.

[0033] In certain embodiments, the method of predicting the rate of ALS progression to disease endpoints in a subject having or suspected of having ALS further comprises: (d) administering or withholding a first therapeutic regimen to the subject if the subject is a slow ALS progressor, and / or (e) administering a second therapeutic regimen to the subject if the subject is a rapid ALS progressor.

[0034] In certain embodiments, determining a level of 4-HNE comprises utilizing an assay comprising a solid surface having an antibody or an antigen-binding portion thereof that binds to a 4-HNE epitope.

[0035] In certain embodiments, the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL.

[0036] In certain embodiments, the level of the 4-HNE in (c)(i) correlates with an ALSFRS- R rate of decline less than about one point per month. In certain embodiments, the level of the 4- HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about 0.7 points per month.

[0037] In certain embodiments, the level of the 4-HNE in (c)(ii) correlates with an ALSFRS- R rate of decline greater than about one point per month. In certain embodiments, the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about 1.2 points per month.

[0038] In certain embodiments, the first therapeutic regimen and the second therapeutic regimen comprise an ALS therapy. In certain embodiments, the first therapeutic regimen and the second therapeutic regimen comprise the same ALS therapy. In certain embodiments, the first NAI-1540949621v1 5Atty. Docket No.14678-018-228 therapeutic regimen and the second therapeutic regimen comprise different ALS therapies.

[0039] In certain embodiments, the second therapeutic regimen comprises a palliative care therapeutic regimen.

[0040] In certain embodiments, the second therapeutic regimen comprises a higher dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen. In certain embodiments, the second therapeutic regimen comprises a lower dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

[0041] In certain embodiments, the second therapeutic regimen comprises administering a dosage of the ALS therapy with increased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen. In certain embodiments, the second therapeutic regimen comprises administering a dosage of the ALS therapy with decreased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

[0042] In certain embodiments, the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol (e.g., Relyvrio, AMX0035), edaravone, riluzole, interleukin-2 (IL- 2), abatacept (CTLA-4 Ig), or any combination thereof. In certain embodiments, the IL-2 is an IL-2 conjugate or an IL-2 mutein. In certain embodiments, the ALS therapy comprises an IL- 2 / abatacept combination therapy. In certain embodiments, the ALS therapy comprises a low dose of IL-2. In certain embodiments, the low dose of IL-2 comprises a dose of IL-2 less than about 5.0 ×106IU / m2 / day. In certain embodiments, the IL-2 is a human recombinant IL 2. In certain embodiments, the IL-2 is aldesleukin.

[0043] In certain embodiments, the first ALS therapy or the second ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol (e.g., Relyvrio, AMX0035), edaravone, riluzole, IL-2, abatacept (CTLA-4 Ig), or any combination thereof.

[0044] In certain embodiments, palliative care comprises management of one or more symptoms of ALS.

[0045] In another aspect, the present disclosure provides a method of treating ALS, comprising administering to a subject predicted to be a short-term ALS survivor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a short-term ALS survivor in that serum or plasma collected from the subject comprises a 4-HNE level greater than a 4-HNE reference range. NAI-1540949621v1 6Atty. Docket No.14678-018-228

[0046] In another aspect, the present disclosure provides a method of treating ALS, comprising administering to a subject predicted to be a long-term ALS survivor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a long-term ALS survivor in that serum or plasma collected from the subject comprises a 4-HNE level equal to or less than a 4-HNE reference range.

[0047] In certain embodiments, determining the level of 4-HNE comprises an assay that uses a composition comprising a solid surface having an antibody or an antigen-binding portion thereof that binds to a 4-HNE epitope. In certain embodiments, the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL.

[0048] In certain embodiments, the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol (e.g., Relyvrio, AMX0035), edaravone, riluzole, IL-2, abatacept (CTLA-4 Ig), or any combination thereof.

[0049] In some embodiments, the present disclosure provides a method of treating ALS comprising: (a) determining whether a biological sample, e.g., a serum or plasma sample, collected from a subject comprises a 4-HNE level greater than a 4-HNE reference range; and (b) if the serum collected from the subject comprises 4-HNE level greater than the 4-HNE reference range, administer a therapeutic regimen. In particular embodiments, the therapeutic regimen is an ALS therapy. In particular embodiments, the therapeutic regimen is palliative care.

[0050] In some embodiments, the present disclosure provides a method of treating ALS comprising: (a) determining whether a biological sample, e.g., a serum or plasma sample, collected from a subject comprises a 4-HNE level equal to or lower than a 4-HNE reference range; and (b) if the serum collected from the subject comprises a 4-HNE level equal to or lower than the 4-HNE reference range, administer a therapeutically effective amount of an ALS therapy to the subject. In particular embodiments, the therapeutic regimen is an ALS therapy.

[0051] In some embodiments, the present disclosure provides a method of treating ALS comprising: (a) determining a 4-HNE level in a biological sample, e.g., a serum or plasma sample, collected from a subject; and (b) administering a first therapeutic regimen if the 4-HNE level in the biological sample is equal to or lower than a 4-HNE reference range; or administering a second therapeutic regimen and / or withholding the first regimen if the 4-HNE level in the biological sample is greater than a 4-HNE reference range. In particular embodiments, the first and / or second therapeutic regimen is an ALS therapy. In particular NAI-1540949621v1 7Atty. Docket No.14678-018-228 embodiments, the first and / or second therapeutic regimen is palliative care.

[0052] In another aspect, the present disclosure provides a kit for detecting and quantifying the level of 4-hydroxynonenal (4-HNE) in a serum or plasma sample obtained from a subject having or suspected of having ALS, wherein the kit comprises a solid surface having an antibody or a fragment thereof comprising a binding epitope for 4-HNE.

[0053] In certain embodiments, the kit further comprises at least one reagent for detecting formation of a 4-HNE-antibody complex.

[0054] In certain embodiments, the kit of the present disclosure is for use in predicting a survival outcome in the subject having or suspected of having ALS.

[0055] In certain embodiments, the kit of the present disclosure is for use in monitoring changes in a survival outcome during the course of or after completing an ALS therapy regimen.

[0056] In another aspect, the present disclosure provides a method of predicting clinical benefit of an ALS therapy comprising: (a) administering the ALS therapy to a subject; (b) collecting a plasma or serum sample from the subject after administering the ALS therapy; (c) determining the level of 4-HNE in the sample collected from the subject, wherein the level of 4- HNE in the sample acts as a surrogate endpoint; (d) predicting the clinical benefit of the ALS therapy as: (i) favorable if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) unfavorable if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE.

[0057] In certain embodiments, a favorable clinical benefit of the ALS therapy is correlated with slow disease progression.

[0058] In certain embodiments, an unfavorable clinical benefit of the ALS therapy is correlated with rapid disease progression.

[0059] In certain embodiments, an unfavorable clinical benefit of the ALS therapy is predictive of death.

[0060] In certain embodiments, the subject is not administered the ALS therapy after the level of the 4-HNE in the sample is determined to be greater than the reference range for 4-HNE.

[0061] In certain embodiments, the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL.

[0062] In certain embodiments, the clinical benefit of the ALS therapy is unknown prior to administration. NAI-1540949621v1 8Atty. Docket No.14678-018-228

[0063] In certain embodiments, use of the level of 4-HNE in the sample as surrogate endpoint can predict clinical benefit in a clinical trial of at least one investigational therapy for ALS. 5.1 Illustrative Embodiments

[0064] Illustrative embodiments of the present disclosure are provided in the paragraphs below: 1. A method of predicting survival in a subject having or suspected of having amyotrophic lateral sclerosis (ALS), comprising: (a) determining a level of 4-hydroxynonenal (4-HNE) in a plasma or serum sample from the subject; and (b) diagnosing the subject as: (i) a long-term ALS survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short-term ALS survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE. 2. A method of predicting survival in a subject having or suspected of having amyotrophic lateral sclerosis (ALS), comprising: (a) obtaining or having obtained a plasma or serum sample from the subject; (b) determining a level of 4-hydroxynonenal (4-HNE) in the subject; and (c) diagnosing the subject as: (i) a long-term survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short-term survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE. 3. The method of embodiment 1 or 2, wherein the method further comprises: (d) administering: (i) a first therapeutic regimen to the subject if the subject is a long-term survivor, or (ii) a second therapeutic regimen to the subject if the subject is a short-term survivor. 4. The method of any one of embodiments 1 to 3, wherein determining the level of 4-HNE NAI-1540949621v1 9Atty. Docket No.14678-018-228 comprises an assay that uses a composition comprising a solid surface having an antibody or an antigen-binding portion thereof that binds to a 4-HNE epitope. 5. The method of any one of embodiments 1 to 4, wherein the level of the 4-HNE in the sample equal to or lower than a reference range for 4-HNE correlates to an increased mean survival time for the subject. 6. The method of any one of embodiments 1 to 4, wherein a level of the 4-HNE in the sample greater than the reference range for 4-HNE correlates to a decreased mean survival time for the subject. 7. The method of any one of embodiments 1 to 4, wherein the level of the 4-HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about one point per month. 8. The method of any one of embodiments 1 to 4, wherein the level of the 4-HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about 0.7 points per month. 9. The method of any one of embodiments 1 to 4, wherein the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about one point per month. 10. The method of any one of embodiments 1 to 4, wherein the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about 1.2 points per month. 11. The method of any one of embodiments 1 to 10, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL. 12. The method of any one of embodiments 3 to 11, wherein the first therapeutic regimen and the second therapeutic regimen comprise an ALS therapy. 13. The method of embodiment 12, wherein the first therapeutic regimen and the second therapeutic regimen comprise the same ALS therapy. 14. The method of embodiment 12, wherein the first therapeutic regimen and the second therapeutic regimen comprise different ALS therapies. 15. The method of any one of embodiments 3 to 12, wherein the second therapeutic regimen comprises a palliative care therapeutic regimen. 16. The method of embodiment 13, wherein the second therapeutic regimen comprises a higher dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen. 17. The method of embodiment 13, wherein the second therapeutic regimen comprises a lower dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the NAI-1540949621v1 10Atty. Docket No.14678-018-228 first therapeutic regimen. 18. The method of embodiment 13, wherein the second therapeutic regimen comprises administering a dosage of the ALS therapy with increased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen. 19. The method of embodiment 13, wherein the second therapeutic regimen comprises administering a dosage of the ALS therapy with decreased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen. 20. The method of embodiment 12, wherein the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, interleukin-2 (IL-2), abatacept (CTLA- 4 Ig), or any combination thereof. 21. The method of embodiment 20, wherein the IL-2 is an IL-2 conjugate or an IL-2 mutein. 22. The method of embodiment 20, wherein the ALS therapy comprises an IL-2 / abatacept combination therapy. 23. The method of any one of embodiments 20 to 22, wherein the ALS therapy comprises a low dose of IL-2. 24. The method of embodiment 23, wherein the low dose of IL-2 comprises a dose of IL-2 less than about 5.0 ×106IU / m2 / day. 25. The method of any one of embodiments 20 to 24, wherein the IL-2 is a human recombinant IL-2. 26. The method of any one of embodiments 20 to 25, wherein the IL-2 is aldesleukin. 27. The method of embodiment 14, wherein the first ALS therapy or the second ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, IL-2, abatacept (CTLA-4 Ig), or any combination thereof. 28. The method of embodiment 15, wherein palliative care comprises management of one or more symptoms of ALS. 29. The method of embodiment 1, wherein the sample was obtained from the subject at ALS diagnosis. 30. A method of determining impact of an ALS therapy on survival of a subject having or suspected of having ALS, comprising: (a) determining a level of 4-hydroxynonenal (4-HNE) in a plasma or serum sample from the subject; NAI-1540949621v1 11Atty. Docket No.14678-018-228 (b) diagnosing the subject as: (i) a long-term ALS survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short-term ALS survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE; (c) administering an ALS therapy to the subject; (d) determining a level of 4-HNE in a plasma or serum sample from the subject following (c); and (e) comparing the level of 4-HNE in (a) to the 4-HNE level in (d), thereby determining the impact of the ALS therapy on survival of the subject. 31. The method of embodiment 30, wherein determining the level of 4-HNE comprises an assay that uses a composition comprising a solid surface having an antibody or an antigen- binding portion thereof that binds to a 4-HNE epitope. 32. The method of embodiment 30 or 31, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL. 33. The method of any one of embodiments 30 to 32, wherein the level of the 4-HNE in the sample equal to or lower than a reference range for 4-HNE correlates to an increased mean survival time for the subject. 34. The method of any one of embodiments 30 to 32, wherein the level of the 4-HNE in the sample greater than the reference range for 4-HNE correlates to a decreased mean survival time for the subject. 35. The method of any one of embodiments 30 to 34, wherein the ALS therapy is predicted to improve survival of the subject when the 4-HNE level in (d) is equal to or less than the level of 4-HNE in (a). 36. The method of any one of embodiments to 35, wherein the ALS therapy is predicted to have no effect or worsen survival of the subject when the 4-HNE level in (d) is higher than the level of 4-HNE in (a). 37. The method of either embodiment 35 or 36, wherein the method further comprises: (f) administering: (i) a continued dosage of the ALS therapy administered in (c) to the subject if the ALS therapy improves survival of the subject, or NAI-1540949621v1 12Atty. Docket No.14678-018-228 (ii) a second therapeutic regimen to the subject if the ALS therapy has no effect or worsens survival of the subject. 38. The method of any one of embodiments 30 to 37, wherein the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, interleukin-2 (IL-2), abatacept (CTLA-4 Ig), or any combination thereof. 39. The method of embodiment 38, wherein the IL-2 is an IL-2 conjugate or an IL-2 mutein. 40. The method of embodiment 38, wherein the ALS therapy comprises an IL-2 / abatacept combination therapy. 41. The method of any one of embodiments 38 to 40, wherein the ALS therapy comprises a low dose of IL-2. 42. The method of embodiment 41, wherein the low dose of IL-2 comprises a dose of IL-2 less than about 5.0 ×106IU / m2 / day. 43. The method of any one of embodiments 38 to 42, wherein the IL-2 is a human recombinant IL-2. 44. The method of any one of embodiments 38 to 43, wherein the IL-2 is aldesleukin. 45. The method of embodiment 37, wherein the second therapeutic regimen comprises a palliative care therapeutic regimen. 46. The method of embodiment 45, wherein palliative care comprises management of one or more symptoms of ALS. 47. The method of any one of embodiments 38 to 46, wherein the subject is naïve to ALS therapy. 48. The method of any one of embodiments 38 to 46, wherein the subject has previously received at least one ALS therapy. 49. A method of predicting the rate of ALS progression to disease endpoints in a subject having or suspected of having ALS, comprising: (a) obtaining or having obtained a plasma or serum sample from the subject; (b) determining a level of 4-hydroxynonenal (4-HNE) in a plasma or serum sample from the subject; and (c) diagnosing the subject as: (i) a slow ALS progressor if the level of the 4-HNE in the sample is equal to or lower than a reference range; or NAI-1540949621v1 13Atty. Docket No.14678-018-228 (ii) a rapid ALS progressor if the level of the 4-HNE in the sample is greater than the reference range, wherein the disease endpoints are death or a need for implementation of invasive respiratory measures. 50. The method of embodiment 49, wherein the method further comprises: (d) administering or withholding a first therapeutic regimen to the subject if the subject is a slow ALS progressor, and / or (e) administering a second therapeutic regimen to the subject if the subject is a rapid ALS progressor. 51. The method of embodiment 49 or 50, wherein determining the level of 4-HNE comprises an assay that uses a composition comprising a solid surface having an antibody or an antigen- binding portion thereof that binds to a 4-HNE epitope. 52. The method of any one of embodiments 49 to 51, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL. 53. The method of any one of embodiments 49 to 52, wherein the level of the 4-HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about one point per month. 54. The method of any one of embodiments 49 to 52, wherein the level of the 4-HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about 0.7 points per month. 55. The method of any one of embodiments 49 to 52, wherein the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about one point per month. 56. The method of any one of embodiments 49 to 52, wherein the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about 1.2 points per month. 57. The method of any one of embodiments 50 to 56, wherein the first therapeutic regimen and the second therapeutic regimen comprise an ALS therapy. 58. The method of embodiment 57, wherein the first therapeutic regimen and the second therapeutic regimen comprise the same ALS therapy. 59. The method of embodiment 57, wherein the first therapeutic regimen and the second therapeutic regimen comprise different ALS therapies. 60. The method of any one of embodiments 50 to 57, wherein the second therapeutic regimen comprises a palliative care therapeutic regimen. 61. The method of embodiment 58, wherein the second therapeutic regimen comprises a higher dosage of the ALS therapy compared to the dosage of the ALS therapy administered in NAI-1540949621v1 14Atty. Docket No.14678-018-228 the first therapeutic regimen. 62. The method of embodiment 58, wherein the second therapeutic regimen comprises a lower dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen. 63. The method of embodiment 58, wherein the second therapeutic regimen comprises administering a dosage of the ALS therapy with increased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen. 64. The method of embodiment 58, wherein the second therapeutic regimen comprises administering a dosage of the ALS therapy with decreased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen. 65. The method of embodiment 57, wherein the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, interleukin-2 (IL-2), abatacept (CTLA- 4 Ig), or any combination thereof. 66. The method of embodiment 65, wherein the IL-2 is an IL-2 conjugate or an IL-2 mutein. 67. The method of embodiment 65, wherein the ALS therapy comprises an IL-2 / abatacept combination therapy. 68. The method of any one of embodiments 65 to 67, wherein the ALS therapy comprises a low dose of IL-2. 69. The method of embodiment 68, wherein the low dose of IL-2 comprises a dose of IL-2 less than about 5.0 ×106IU / m2 / day. 70. The method of any one of embodiments 65 to 69, wherein the IL-2 is a human recombinant IL-2. 71. The method of any one of embodiments 65 to 70, wherein the IL-2 is aldesleukin. 72. The method of embodiment 59, wherein the first ALS therapy or the second ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, IL-2, abatacept (CTLA-4 Ig), or any combination thereof. 73. The method of embodiment 60, wherein palliative care comprises management of one or more symptoms of ALS. 74. A method of treating ALS, comprising administering to a subject predicted to be a short- term ALS survivor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a short-term ALS survivor in that serum or plasma collected from the subject NAI-1540949621v1 15Atty. Docket No.14678-018-228 comprises a 4-HNE level greater than a 4-HNE reference range. 75. A method of treating ALS, comprising administering to a subject predicted to be a long- term ALS survivor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a long-term ALS survivor in that serum or plasma collected from the subject comprises a 4-HNE level equal to or less than a 4-HNE reference range. 76. The method of embodiment 74 or 75, wherein determining a 4-HNE level comprises an assay comprising a solid surface having an antibody or an antigen-binding portion thereof that binds to a 4-HNE epitope. 77. The method of any one of embodiments 74 to 76, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL. 78. The method of any one of embodiments 74 to 77, wherein the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, IL-2, abatacept (CTLA-4 Ig), or any combination thereof. 79. A kit for detecting and quantifying the level of 4-hydroxynonenal (4-HNE) in a serum or plasma sample obtained from a subject having or suspected of having ALS, wherein the kit comprises a solid surface having an antibody or a fragment thereof comprising a binding epitope for 4-HNE. 80. The kit of embodiment 79, further comprising at least one reagent for detecting formation of a 4-HNE-antibody complex. 81. The kit of embodiment 79 or embodiment 80 for use in predicting a survival outcome in the subject having or suspected of having ALS. 82. The kit of any one of embodiments 79 to 81 for use in monitoring changes in a survival outcome during the course of or after completing an ALS therapy regimen. 83. A method of predicting clinical benefit of an ALS therapy comprising: (a) administering the ALS therapy to a subject; (b) collecting a plasma or serum sample from the subject after administering the ALS therapy; (c) determining the level of 4-HNE in the sample collected from the subject, wherein the level of 4-HNE in the sample acts as a surrogate endpoint; (d) predicting the clinical benefit of the ALS therapy as: (i) favorable if the level of the 4-HNE in the sample is equal to or lower than NAI-1540949621v1 16Atty. Docket No.14678-018-228 a reference range for 4-HNE; or (ii) unfavorable if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE. 84. The method of embodiment 83, wherein a favorable clinical benefit of the ALS therapy is correlated with slow disease progression. 85. The method of embodiment 83, wherein an unfavorable clinical benefit of the ALS therapy is correlated with rapid disease progression. 86. The method of embodiment 83 or 85, wherein an unfavorable clinical benefit of the ALS therapy is predictive of death. 87. The method of embodiment 83, wherein the subject is not administered the ALS therapy after the level of the 4-HNE in the sample is determined to be greater than the reference range for 4-HNE. 88. The method of any one of embodiments 83 to 87, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL. 89. The method of any one of embodiments 83 to 88, wherein the clinical benefit of the ALS therapy is unknown prior to administration. 90. The method of any one of embodiments 83 to 89, wherein use of the level of 4-HNE in the sample as surrogate endpoint can predict clinical benefit in a clinical trial of at least one investigational therapy for ALS. 6. BRIEF DESCRIPTION OF THE FIGURES

[0065] The foregoing and other objects, features, and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

[0066] FIGS.1A-1D depict graphs showing correlation of ALS disease progression and survival in 50 ALS patients. FIG.1A shows the rate of disease progression (ALS-FRS points / month) correlated with the duration of time (“survival”) from symptom onset to death. FIG.1B shows the rate of disease progression correlated with the duration of time (“survival”) from diagnosis to death. FIG.1C shows the rate of disease progression correlated with the duration of time (“survival”) from date of the last sample collection to death. FIG.1D shows the rate of disease progression correlated with the duration of time (“survival”) from symptom onset NAI-1540949621v1 17Atty. Docket No.14678-018-228 to diagnosis. Correlation was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p < 0.001 and p=0.006). Graphs shown are scatterplots representing 50 ALS patients with a regression line fitted to demonstrate the relationship between disease progression and survival.

[0067] FIGS.2A-2C depict graphs showing correlation of 4-HNE expression and survival in 50 ALS patients. FIG.2A shows the mean 4-HNE expression of all serial samples collected from the 50 ALS patients correlated with the duration of time (“survival”) from onset of disease to death. FIG.2B shows the mean 4-HNE expression of all serial samples collected from the 50 ALS patients correlated with the duration of time (“survival”) from the date of diagnosis to death. FIG.2C shows 4-HNE expression as measured in the last, single sample collected from the 50 ALS patients correlated with the duration of time (“survival”) from the last sample collection date to death. Correlation was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p < 0.001). Graphs shown are scatterplots representing 50 ALS patients with a regression line fitted to demonstrate the relationship between 4-HNE expression and survival.

[0068] FIGS.3A-3C depict graphs showing correlation of 4-HNE expression and survival in 50 ALS patients. FIG.3A shows 4-HNE expression in the first, single sample collected from the 50 ALS patients at diagnosis correlated with the duration of time (“survival”) from onset of ALS disease to death. FIG.3B shows 4-HNE expression in the first, single sample collected from the 50 ALS patients at diagnosis correlated with the duration of time (“survival”) from ALS diagnosis to death. FIG.3C shows 4-HNE expression in the first, single sample collected from the 50 ALS patients at diagnosis correlated with the duration of time occurring between the date of onset of ALS disease to the date of diagnosis of ALS. Correlation was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p < 0.001 and p=0.002). Graphs shown are scatterplots representing 50 ALS patients with a regression line fitted to demonstrate the relationship between 4-HNE expression and survival.

[0069] FIGS.4A-4D depict graphs showing correlation of 4-HNE expression and survival in male (n=31) and female (n=19) ALS patients. FIGS.4A and 4B show 4-HNE expression in the first, single sample collected from the 31 male ALS patients (FIG.4A) and the 19 female ALS patients (FIG.4B) at diagnosis correlated with the duration of time (“survival”) from the onset NAI-1540949621v1 18Atty. Docket No.14678-018-228 of ALS disease to death. FIGS.4C and 4D show 4-HNE expression in the first, single sample collected from the 31 male ALS patients (FIG.4C) and the 19 female ALS patients (FIG.4D) at diagnosis correlated with the duration of time (“survival”) from ALS diagnosis to death. Correlation was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p < 0.001, p=0.050, p=0.013, and p=0.365). Graphs shown are scatterplots representing 31 male ALS patients (FIGS.4A and 4C) or 19 female ALS patients (FIGS.4B and 4D) with a regression line fitted to demonstrate the relationship between 4-HNE expression and survival.

[0070] FIGS.5A-5C depict graphs showing correlation of 4-HNE expression and rate of disease progression in 50 ALS patients. FIG.5A shows the mean 4-HNE expression of all serial samples collected from the 50 ALS patients correlated with the rate of ALS disease progression (ALS-FRS / month). FIG.5B shows 4-HNE expression in the first, single sample collected from the 50 ALS patients at diagnosis correlated with the rate of ALS disease progression. FIG.5C shows 4-HNE expression in the last, single sample collected from the 50 ALS patients before death correlated with the rate of ALS disease progression. Correlation was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p=0.002, p=0.026, and p=0.008). Graphs shown are scatterplots representing 50 ALS patients with a regression line fitted to demonstrate the relationship between 4-HNE expression and the rate of ALS disease progression.

[0071] FIGS.6A and 6B depict graphs showing correlation of the rate of change in 4-HNE expression and the rate of disease progression in 50 ALS patients. FIG.6A shows the average rate of change in 4-HNE levels determined from the concentration change between a single patient sample taken on the first day of the month and a single patient sample taken on the last day of the month. The rate of change in 4-HNE levels per month is then averaged for each individual ALS patient and correlated to the rate of disease progression (i.e., the average change in ALS-FRS score per month). FIG.6B shows the average rate of change of mean 4-HNE expression levels in the 50 ALS patients per month as determined from the average amount of changes in 4-HNE expression as measured between two serial samples assayed within the same month, correlated with the rate of ALS disease progression. Correlation was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p < 0.001 and p =0.003). Graphs shown are scatterplots representing 50 ALS NAI-1540949621v1 19Atty. Docket No.14678-018-228 patients with a regression line fitted to demonstrate the relationship between the rate of 4-HNE expression change and ALS disease progression.

[0072] FIGS.7A and 7B depict graphs showing correlation of the rate of change in 4-HNE expression and survival in 50 ALS patients. FIGS.7A and 7B show the change in 4-HNE serum expression from first 4-HNE expression level assayed at diagnosis to the 4-HNE expression level assayed at last sample collected before death, divided by the duration of time in months between the two sample collections. These changes in 4-HNE serum expression were correlated with the duration of time (in months) between the age of the patient at symptom onset and their age at death (FIG.7A) or with the duration of time (in months) between the age of the patient at ALS diagnosis and their age at death (FIG.7B). Correlation was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p < 0.001). Graphs shown are scatterplots representing the 50 ALS patients with a regression line fitted to demonstrate the relationship between the rate of 4-HNE expression change and survival.

[0073] FIGS.8A and 8B depict graphs showing measurements of 4-HNE and ox-LDL levels in serum samples collected from 50 ALS patients and 53 healthy control subjects. FIG. 8A shows mean 4-HNE expression in 50 ALS patients compared with mean 4-HNE expression in 53 healthy controls. FIG.8B shows mean ox-LDL expression in 50 ALS patients compared with mean ox-LDL expression in 53 healthy controls. Comparisons were performed using a Student’s t-test presented with a p value (p < 0.001).

[0074] FIG.9 depicts a graph showing correlation of 4-HNE and ox-LDL levels measured in all serial samples (n=298) collected from 50 ALS patients. Correlation between 4-HNE and ox- LDL serum expression was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p < 0.001). Graph shown is a scatterplot of each sample with a regression line fitted to demonstrate the relationship between ox-LDL and 4-HNE.

[0075] FIGS.10A-10F depict graphs showing correlation of ox-LDL expression and survival in 50 ALS patients. FIG.10A shows the mean ox-LDL expression of all serial samples collected from the 50 ALS patients correlated with the duration of time (“survival”) from onset of disease to death. FIG.10B shows the mean ox-LDL expression of all serial samples collected from the 50 ALS patients correlated with the duration of time (“survival”) from date of diagnosis to death. FIG.10C shows ox-LDL expression in the last, single sample collected from the 50 NAI-1540949621v1 20Atty. Docket No.14678-018-228 ALS patients correlated with the duration of time (“survival”) from last sample collection date to death. FIG.10D shows ox-LDL expression in the first, single sample collected from the 50 ALS patients at diagnosis correlated with the duration of time (“survival”) from onset of ALS disease to death. FIG.10E shows ox-LDL expression in the first, single sample collected from the 50 ALS patients at diagnosis correlated with the duration of time (“survival”) from ALS diagnosis to death. FIG.10F shows ox-LDL expression in the first, single sample collected from the 50 ALS patients at diagnosis correlated with the duration of time occurring between the date of onset of ALS disease to the date of diagnosis of ALS. Correlation was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p=0.646, p=0.222, p=0.078, p=0.724, p=0.209, and p=0.592). Graphs shown are scatterplots representing 50 ALS patients with a regression line fitted to demonstrate the relationship between ox-LDL expression and survival.

[0076] FIGS.11A-11C depict graphs showing correlation of ox-LDL expression and disease progression in 50 ALS patients. FIG.11A shows the mean ox-LDL expression of all serial samples collected from the 50 ALS patients correlated with rate of ALS disease progression (ALS-FRS / month). FIG.11B shows ox-LDL expression in the first, single sample collected from the 50 ALS patients at diagnosis correlated with rate of ALS disease progression. FIG. 11C shows ox-LDL expression in the last, single sample collected from the 50 ALS patients before death correlated with rate of ALS disease progression. Correlation was determined using Spearman Rank Order in SigmaStat software and presented with the correlation coefficient r (rho) and p value (p=0.279, p=0.793, and p=0.133). Graphs shown are scatterplots representing 50 ALS patients with a regression line fitted to demonstrate the relationship between ox-LDL expression and ALS disease progression. 7. DETAILED DESCRIPTION

[0077] In certain aspects, the present disclosure provides methods for predicting survival in a subject having or suspected of having amyotrophic lateral sclerosis (ALS), and associated methods of treating ALS. In certain aspects, presented herein are methods for determining impact of an ALS therapy on survival of a subject having or suspected of having ALS, and associated methods of treating ALS. In certain aspects, presented herein are methods of predicting the rate of ALS progression in a subject having or suspected of having ALS, and associated methods of treating ALS. NAI-1540949621v1 21Atty. Docket No.14678-018-228

[0078] Generally, the methods described herein comprise determining a level of 4- hydroxynonenal (4-HNE) in biological sample from a subject (e.g., a blood, serum or plasma sample). In particular embodiments, the level of 4-HNE diagnoses the subject as a long-term ALS survivor or a short-term ALS survivor. For example, in certain embodiments the level of 4- HNE in the sample subject is equal to or lower than a reference range for 4-HNE and the subject us diagnosed as a long-term ALS survivor. In certain embodiments the level of 4-HNE in the sample subject is greater than the reference range for 4-HNE and the subject us diagnosed as a short-term ALS survivor. Unless otherwise noted, “4-HNE level” and “4-HNE-expression” are used interchangeably herein.

[0079] A person skilled in the art would understand that the methods provided herein include all permutations, including modifications, and combinations of ALS therapies disclosed herein, patients, dosing regimens, diagnostic and ALS staging criteria, and therapeutic outcomes as described above and below.

[0080] As used in this disclosure including the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. The terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein unless the context clearly dictates otherwise.

[0081] The term “about” means within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less variation of a given value or range. As used herein, a phrase such as “about A,” includes and is deemed to also recite “A.” As used herein, a phrase such as “about A to about B” includes and is deemed to also recite “A to B.”

[0082] The term “between” as used in a phrase as such “between A and B” or “between A- B” refers to a range including both A and B. 7.1 Methods of Predicting Survival

[0083] In certain aspects, presented herein are methods for predicting survival in a subject having or suspected of having ALS. “Subjects” and “patients” are used synonymously herein and can refer to one or more of the patient populations described in Section 7.7.

[0084] In certain embodiments, a method for predicting survival in a subject having or suspected of having ALS comprises (a) determining a level of 4-HNE in biological sample from a subject, for example, in a plasma or serum sample from the subject, and (b) diagnosing the NAI-1540949621v1 22Atty. Docket No.14678-018-228 subject as: (i) a long-term ALS survivor if the level of 4-HNE in the sample is equal to or lower than a reference range for 4-HNE, or (ii) a short-term ALS survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE. In certain embodiments, a method for predicting survival in a subject having or suspected of having ALS comprises (a) obtaining or having obtained a biological sample from the subject, (b) determining a level of 4-HNE in biological sample from a subject, for example, in a plasma or serum sample from the subject, and (c) diagnosing the subject as: (i) a long-term ALS survivor if the level of 4-HNE in the sample is equal to or lower than a reference range for 4-HNE, or (ii) a short-term ALS survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE. In particular embodiments, a method of predicting survival in a subject having or suspected of having ALS may further comprise (d) administering (i) a first therapeutic regimen to the subject if the subject is a long-term survivor, or (ii) a second therapeutic regimen to the subject if the subject is a short-term survivor.

[0085] In certain aspects, provided herein are methods of determining impact of an ALS therapy on survival of a subject having or suspected of having ALS, comprising: (a) determining a level of 4-HNE in a sample, e.g., a plasma or serum sample, from the subject; (b) diagnosing the subject as: (i) a long-term ALS survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short-term ALS survivor if the level of the 4- HNE in the sample is greater than the reference range for 4-HNE; (c) administering an ALS therapy to the subject; (d) determining a level of 4-HNE in a plasma or serum sample from the subject following (c); and (e) comparing the level of 4-HNE in (a) to the 4-HNE level in (d), thereby determining the impact of the ALS therapy on survival of the subject.

[0086] In particular embodiments of such methods, the ALS therapy is predicted to improve survival of the subject when the 4-HNE level in (d) is equal to or less than the level of 4-HNE in (a). In particular embodiments of such methods, the ALS therapy is predicted to have no effect or worsen survival of the subject when the 4-HNE level in (d) is greater than the level of 4-HNE in (a). In specific embodiments of such methods, the methods may further comprise administering: (i) a continued dosage of the ALS therapy administered in (c) to the subject if the ALS therapy improves survival of the subject, or (ii) a second therapeutic regimen to the subject if the ALS therapy has no effect or worsens survival of the subject.

[0087] In certain aspects, provided herein are methods of predicting the rate of ALS NAI-1540949621v1 23Atty. Docket No.14678-018-228 progression to disease endpoints in a subject having or suspected of having ALS, comprising: (a) obtaining or having obtained a sample, e.g., a plasma or serum sample, from the subject; (b)determining a level of 4-HNE in a plasma or serum sample from the subject; and (c) diagnosing the subject as: (i) a slow ALS progressor if the level of the 4-HNE in the sample is equal to or lower than a 4-HNE reference range; or (ii) a rapid ALS progressor if the level of the 4-HNE in the sample is greater than the 4-HNE reference range, wherein the disease endpoints are death or a need for implementation of invasive respiratory measures. In particular embodiments, such methods may further comprise (d) administering or withholding a first therapeutic regimen to the subject if the subject is a slow ALS progressor, and / or (e) administering a second therapeutic regimen to the subject if the subject is a rapid ALS progressor.

[0088] Methods for determining a level of 4-HNE in a biological sample are well known. Exemplary methods of determining a level of 4-HNE are presented in Section 7.2. In particular embodiments, determining a level of 4-HNE comprises utilization of an assay, e.g., an ELISA assay, that uses a composition comprising a solid surface having an antibody or an antigen- binding portion thereof that binds to a 4-HNE epitope.

[0089] 4-HNE Reference Range. In certain embodiments, a reference range for 4-HNE can range from about 0.1 µg / mL to about 20 µg / mL, about 0.15 µg / mL to about 19 µg / mL, about 0.2 µg / mL to about 18 µg / mL, about 0.25 µg / mL to about 17 µg / mL, about 0.3 µg / mL to about 16 µg / mL, about 0.35 µg / mL to about 15 µg / mL, about 0.4 µg / mL to about 14 µg / mL, about 0.45 µg / mL to about 13 µg / mL, or about 0.5 µg / mL to about 12 µg / mL. In particular embodiments, a reference range for 4-HNE can range from about 0.5 µg / mL to about 12 µg / mL. In particular embodiments, a reference range for 4-HNE can range from about 0.5 µg / mL to about 13 µg / mL. In particular embodiments, a reference range for 4-HNE can range from about 0.5 µg / mL to about 14 µg / mL. In particular embodiments, a reference range for 4-HNE can range from about 0.5 µg / mL to about 15 µg / mL.

[0090] In some embodiments, a reference range for 4-HNE can range from about 0.5 µg / mL to about 20 µg / mL, about 0.5 µg / mL to about 19 µg / mL, about 0.5 µg / mL to about 18 µg / mL, about 0.5 µg / mL to about 17 µg / mL, about 0.5 µg / mL to about 16 µg / mL, about 0.5 µg / mL to about 15 µg / mL, about 0.5 µg / mL to about 14 µg / mL, about 0.5 µg / mL to about 13 µg / mL, about 0.5 µg / mL to about 12 µg / mL, about 0.5 µg / mL to about 10 µg / mL, about 0.5 µg / mL to NAI-1540949621v1 24Atty. Docket No.14678-018-228 about 9 µg / mL, about 0.5 µg / mL to about 8 µg / mL, about 0.5 µg / mL to about 7 µg / mL, about 0.5 µg / mL to about 6 µg / mL, about 0.5 µg / mL to about 5 µg / mL, about 0.5 µg / mL to about 3 µg / mL, about 0.5 µg / mL to about 2 µg / mL, or about 0.5 µg / mL to about 1 µg / mL.

[0091] In some embodiments, a reference range for 4-HNE can range from about 0.1 µg / mL to about 12 µg / mL, about 0.2 µg / mL to about 12 µg / mL, about 0.3 µg / mL to about 12 µg / mL, about 0.4 µg / mL to about 12 µg / mL, about 0.5 µg / mL to about 12 µg / mL, about 0.6 µg / mL to about 12 µg / mL, about 0.7 µg / mL to about 12 µg / mL, about 0.8 µg / mL to about 12 µg / mL, about 0.9 µg / mL to about 12 µg / mL, about 1 µg / mL to about 12 µg / mL, about 2 µg / mL to about 12 µg / mL, about 3 µg / mL to about 12 µg / mL, about 4 µg / mL to about 12 µg / mL, about 5 µg / mL to about 12 µg / mL, about 6 µg / mL to about 12 µg / mL, about 7 µg / mL to about 12 µg / mL, about 8 µg / mL to about 12 µg / mL, about 9 µg / mL to about 12 µg / mL, or about 10 µg / mL to about 12 µg / mL.

[0092] In some embodiments, a reference range for 4-HNE can range from about 0.1 µg / mL to about 13 µg / mL, about 0.2 µg / mL to about 13 µg / mL, about 0.3 µg / mL to about 13 µg / mL, about 0.4 µg / mL to about 13 µg / mL, about 0.5 µg / mL to about 13 µg / mL, about 0.6 µg / mL to about 13 µg / mL, about 0.7 µg / mL to about 13 µg / mL, about 0.8 µg / mL to about 13 µg / mL, about 0.9 µg / mL to about 13 µg / mL, about 1 µg / mL to about 13 µg / mL, about 2 µg / mL to about 13 µg / mL, about 3 µg / mL to about 13 µg / mL, about 4 µg / mL to about 13 µg / mL, about 5 µg / mL to about 13 µg / mL, about 6 µg / mL to about 13 µg / mL, about 7 µg / mL to about 13 µg / mL, about 8 µg / mL to about 13 µg / mL, about 9 µg / mL to about 13 µg / mL, or about 10 µg / mL to about 13 µg / mL.

[0093] In some embodiments, a reference range for 4-HNE can range from about 0.1 µg / mL to about 14 µg / mL, about 0.2 µg / mL to about 14 µg / mL, about 0.3 µg / mL to about 14 µg / mL, about 0.4 µg / mL to about 14 µg / mL, about 0.5 µg / mL to about 14 µg / mL, about 0.6 µg / mL to about 14 µg / mL, about 0.7 µg / mL to about 14 µg / mL, about 0.8 µg / mL to about 14 µg / mL, about 0.9 µg / mL to about 14 µg / mL, about 1 µg / mL to about 14 µg / mL, about 2 µg / mL to about 14 µg / mL, about 3 µg / mL to about 14 µg / mL, about 4 µg / mL to about 14 µg / mL, about 5 µg / mL to about 14 µg / mL, about 6 µg / mL to about 14 µg / mL, about 7 µg / mL to about 14 µg / mL, about 8 µg / mL to about 14 µg / mL, about 9 µg / mL to about 14 µg / mL, or about 10 µg / mL to about 14 µg / mL.

[0094] In some embodiments, a reference range for 4-HNE can range from about 0.1 µg / mL NAI-1540949621v1 25Atty. Docket No.14678-018-228 to about 15 µg / mL, about 0.2 µg / mL to about 15 µg / mL, about 0.3 µg / mL to about 15 µg / mL, about 0.4 µg / mL to about 15 µg / mL, about 0.5 µg / mL to about 15 µg / mL, about 0.6 µg / mL to about 15 µg / mL, about 0.7 µg / mL to about 15 µg / mL, about 0.8 µg / mL to about 15 µg / mL, about 0.9 µg / mL to about 15 µg / mL, about 1 µg / mL to about 15 µg / mL, about 2 µg / mL to about 15 µg / mL, about 3 µg / mL to about 15 µg / mL, about 4 µg / mL to about 15 µg / mL, about 5 µg / mL to about 15 µg / mL, about 6 µg / mL to about 15 µg / mL, about 7 µg / mL to about 15 µg / mL, about 8 µg / mL to about 15 µg / mL, about 9 µg / mL to about 15 µg / mL, or about 10 µg / mL to about 15 µg / mL.

[0095] In some embodiments, a reference range for 4-HNE can range from about 0.5 µg / mL to about 15 µg / mL, from about 0.5 µg / mL to about 14 µg / mL, from about 0.5 µg / mL to about 13 µg / mL, from about 0.5 µg / mL to about 12 µg / mL, from about 0.5 µg / mL to about 11 µg / mL, from about 0.5 µg / mL to about 10 µg / mL, from about 0.5 µg / mL to about 9 µg / mL, from about 0.5 µg / mL to about 8 µg / mL, from about 0.5 µg / mL to about 7 µg / mL, from about 0.5 µg / mL to about 6 µg / mL, from about 0.5 µg / mL to about 5 µg / mL, from about 0.5 µg / mL to about 4 µg / mL, from about 0.5 µg / mL to about 3 µg / mL, from about 0.5 µg / mL to about 2 µg / mL, or from about 0.5 µg / mL to about 1 µg / mL.

[0096] In some embodiments, a reference range for 4-HNE can range from about 1 µg / mL to about 15 µg / mL, from about 1 µg / mL to about 14 µg / mL, from about 1 µg / mL to about 13 µg / mL, from about 1 µg / mL to about 12 µg / mL, from about 1 µg / mL to about 11 µg / mL, from about 1 µg / mL to about 10 µg / mL, from about 1 µg / mL to about 9 µg / mL, from about 1 µg / mL to about 8 µg / mL, from about 1 µg / mL to about 7 µg / mL, from about 1 µg / mL to about 6 µg / mL, from about 1 µg / mL to about 5 µg / mL, from about 1 µg / mL to about 4 µg / mL, from about 1 µg / mL to about 3 µg / mL, or from about 1 µg / mL to about 2 µg / mL.

[0097] In some embodiments, a reference range for 4-HNE can range from about 2 µg / mL to about 15 µg / mL, from about 2 µg / mL to about 14 µg / mL, from about 2 µg / mL to about 13 µg / mL, from about 2 µg / mL to about 12 µg / mL, from about 2 µg / mL to about 11 µg / mL, from about 2 µg / mL to about 10 µg / mL, from about 2 µg / mL to about 9 µg / mL, from about 2 µg / mL to about 8 µg / mL, from about 2 µg / mL to about 7 µg / mL, from about 2 µg / mL to about 6 µg / mL, from about 2 µg / mL to about 5 µg / mL, from about 2 µg / mL to about 4 µg / mL, or from about 2 µg / mL to about 3 µg / mL.

[0098] In some embodiments, a reference range for 4-HNE can range from about 3 µg / mL to NAI-1540949621v1 26Atty. Docket No.14678-018-228 about 15 µg / mL, from about 3 µg / mL to about 14 µg / mL, from about 3 µg / mL to about 13 µg / mL, from about 3 µg / mL to about 12 µg / mL, from about 3 µg / mL to about 11 µg / mL, from about 3 µg / mL to about 10 µg / mL, from about 3 µg / mL to about 9 µg / mL, from about 3 µg / mL to about 8 µg / mL, from about 3 µg / mL to about 7 µg / mL, from about 3 µg / mL to about 6 µg / mL, from about 3 µg / mL to about 5 µg / mL, or from about 3 µg / mL to about 4 µg / mL.

[0099] In some embodiments, a reference range for 4-HNE can range from about 4 µg / mL to about 15 µg / mL, from about 4 µg / mL to about 14 µg / mL, from about 4 µg / mL to about 13 µg / mL, from about 4 µg / mL to about 12 µg / mL, from about 4 µg / mL to about 11 µg / mL, from about 4 µg / mL to about 10 µg / mL, from about 4 µg / mL to about 9 µg / mL, from about 4 µg / mL to about 8 µg / mL, from about 4 µg / mL to about 7 µg / mL, from about 4 µg / mL to about 6 µg / mL, or from about 4 µg / mL to about 5 µg / mL.

[0100] In some embodiments, a reference range for 4-HNE can range from about 5 µg / mL to about 15 µg / mL, from about 5 µg / mL to about 14 µg / mL, from about 5 µg / mL to about 13 µg / mL, from about 5 µg / mL to about 12 µg / mL, from about 5 µg / mL to about 11 µg / mL, from about 5 µg / mL to about 10 µg / mL, from about 5 µg / mL to about 9 µg / mL, from about 5 µg / mL to about 8 µg / mL, from about 5 µg / mL to about 7 µg / mL, or from about 5 µg / mL to about 6 µg / mL.

[0101] In some embodiments, a reference range for 4-HNE can range from about 6 µg / mL to about 15 µg / mL, from about 6 µg / mL to about 14 µg / mL, from about 6 µg / mL to about 13 µg / mL, from about 6 µg / mL to about 12 µg / mL, from about 6 µg / mL to about 11 µg / mL, from about 6 µg / mL to about 10 µg / mL, from about 6 µg / mL to about 9 µg / mL, from about 6 µg / mL to about 8 µg / mL, or from about 6 µg / mL to about 7 µg / mL.

[0102] In some embodiments, a reference range for 4-HNE can range from about 7 µg / mL to about 15 µg / mL, from about 7 µg / mL to about 14 µg / mL, from about 7 µg / mL to about 13 µg / mL, from about 7 µg / mL to about 12 µg / mL, from about 7 µg / mL to about 11 µg / mL, from about 7 µg / mL to about 10 µg / mL, from about 7 µg / mL to about 9 µg / mL, or from about 7 µg / mL to about 8 µg / mL.

[0103] In some embodiments, a reference range for 4-HNE can range from about 8 µg / mL to about 15 µg / mL, from about 8 µg / mL to about 14 µg / mL, from about 8 µg / mL to about 13 µg / mL, from about 8 µg / mL to about 12 µg / mL, from about 8 µg / mL to about 11 µg / mL, from about 8 µg / mL to about 10 µg / mL, or from about 8 µg / mL to about 9 µg / mL. NAI-1540949621v1 27Atty. Docket No.14678-018-228

[0104] In some embodiments, a reference range for 4-HNE can range from about 9 µg / mL to about 15 µg / mL, from about 9 µg / mL to about 14 µg / mL, from about 9 µg / mL to about 13 µg / mL, from about 9 µg / mL to about 12 µg / mL, from about 9 µg / mL to about 11 µg / mL, from about 9 µg / mL to about 10 µg / mL, from about 10 µg / mL to about 15 µg / mL, from about 10 µg / mL to about 14 µg / mL, from about 10 µg / mL to about 13 µg / mL, from about 10 µg / mL to about 12 µg / mL, from about 10 µg / mL to about 11 µg / mL, from about 11 µg / mL to about 15 µg / mL, from about 11 µg / mL to about 14 µg / mL, from about 11 µg / mL to about 13 µg / mL, from about 11 µg / mL to about 12 µg / mL.

[0105] For ease of description, the term “reference range,” as used herein, unless otherwise noted, may refer to a range of values as well as to a particular value. Thus, the term “4-HNE range,” as used herein, unless otherwise noted, may refer to a range of 4-HNE levels of to a particular 4-HNE level For example, in certain embodiments, a reference range for 4-HNE can be about 0.1 µg / mL, about 0.2 µg / mL, about 0.3 µg / mL, about 0.4 µg / mL, about 0.5 µg / mL, about 0.6 µg / mL, about 0.7 µg / mL, about 0.8 µg / mL, about 0.9 µg / mL, about 1 µg / mL, about 1.5 µg / mL, about 2 µg / mL, about 2.5 µg / mL, about 3 µg / mL, about 3.5 µg / mL, about 4 µg / mL, about 4.5 µg / mL, about 5 µg / mL, about 5.5 µg / mL, about 6 µg / mL, about 6.5 µg / mL, about 7 µg / mL, about 7.5 µg / mL, about 8 µg / mL, about 8.5 µg / mL, about 9 µg / mL, about 9.5 µg / mL, about 10 µg / mL, about 10.5 µg / mL, about 11 µg / mL, about 11.5 µg / mL, about 12 µg / mL, about 12.5 µg / mL, about 13 µg / mL, about 13.5 µg / mL, about 14 µg / mL, about 14.5 µg / mL, about 15 µg / mL, about 15.5 µg / mL, about 16 µg / mL, about 16.5 µg / mL, about 17 µg / mL, about 17.5 µg / mL, about 18 µg / mL, about 18.5 µg / mL, about 19 µg / mL, about 19.5 µg / mL, or about 20 µg / mL. In particular embodiments, a reference range for 4-HNE can be about 3 µg / mL, about 3.5 µg / mL, about 4 µg / mL, about 4.5 µg / mL, about 5 µg / mL, about 5.5 µg / mL, about 6 µg / mL, about 6.5 µg / mL, about 7 µg / mL, about 7.5 µg / mL, or about 8 µg / mL. In particular embodiments, a 4-HNE reference range can be about 5 µg / mL. Long-Term ALS Survivors

[0106] In certain aspects, presented herein are methods for predicting survival in a subject having or suspected of having ALS comprising (a) determining a level of 4-HNE in biological sample from a subject, for example, in a plasma or serum sample from the subject, and (b) diagnosing the subject as a long-term ALS survivor if the level of 4-HNE in the sample is equal to or lower than a reference range for 4-HNE. In certain embodiments, presented herein are NAI-1540949621v1 28Atty. Docket No.14678-018-228 methods for predicting survival in a subject having or suspected of having ALS comprising (a) obtaining or having obtained a biological sample from the subject, (b) determining a level of 4- HNE in biological sample from a subject, for example, in a plasma or serum sample from the subject, and (c) diagnosing the subject as a long-term ALS survivor if the level of 4-HNE in the sample is equal to or lower than a reference range for 4-HNE. In particular embodiments, a method of predicting survival in a subject having or suspected of having ALS may further comprise (d) administering (i) a first therapeutic regimen to the subject if the subject is a long- term survivor.

[0107] A long-term ALS survivor may, for example, be an ALS subject that exhibits a particular ALSFRS-R score rate of decline. ALSFRS-R is a 12-item scale assessing motor impairment with regard to bulbar function, hand and arm function, walking / gross motor abilities, and respiration. The ALSFRS-R includes 12 questions, each being rated on a five-point scale from 0=cannot do, to 4=normal ability. Individual item scores are summed to produce a reported score of between 0=worst and 48=best. See Cedarbaum JM, J Neurol Sci 1999; 169: 13-21. An ALSFRS-R score can be used to determine progression of the ALS disease. For example, the progression may be evaluated via the decline in the number of ALSFRS-R points per month. In certain embodiments, a long-term ALS survivor is an ALS subject that exhibits a decline in ALSFRS-R score of less than about 1.0 to less than about 0.2 points per month (points / month), e.g., less than about 1.0 point / month, less than about 0.9 points / month, less than about 0.8 points / month, less than about 0.7 points / month, less than about 0.6 points / month, less than about 0.5 points / month, less than about 0.4 points / month, less than about 0.3 points / month, or less than about 0.2 points / month. In certain embodiments, a reference range for 4-HNE correlates with such an ALSFRS-R rate of decline as described hereinabove. In certain embodiments, a subject having or suspected of having ALS is diagnosed a long-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject contains a level of 4-HNE equal to or lower than such a reference range for 4-NHE.

[0108] In certain embodiments, the reference range for 4-HNE ranges from about 0.5 µg / mL to about 15 µg / mL, from about 0.5 µg / mL to about 14 µg / mL, from about 0.5 µg / mL to about 13 µg / mL, from about 0.5 µg / mL to about 12 µg / mL, from about 0.5 µg / mL to about 11 µg / mL, or from about 0.5 µg / mL to about 10 µg / mL. In particular embodiments, the reference range for 4- HNE ranges from about 0.5 µg / mL to about 12 µg / mL. In certain embodiments, the reference NAI-1540949621v1 29Atty. Docket No.14678-018-228 range is about 0.5 µg / mL, about 1 µg / mL, about 2 µg / mL, about 3 µg / mL, about 4 µg / mL, about 5 µg / mL, about 6 µg / mL, about 7 µg / mL, about 8 µg / mL, about 9 µg / mL, about 10 µg / mL, 11 µg / mL, about 12 µg / mL, about 13 µg / mL or about 14 µg / mL, 15 µg / mL HNE.

[0109] A long-term ALS survivor may, for example, be an ALS subject that exhibits a particular Appel Amyotrophic Lateral Sclerosis (AALS) scale rate of decline. The AALS scale consists of 5 subscales (Bulbar Function, Respiratory Function, Overall Muscle Strength, Upper Extremity, and Lower Extremity Function) with scores ranging from 30 (Normal) to 164 (Maximal Dysfunction). See Haverkamp, Appel, and Appel. Brain.1995 Jun;118 (Pt 3):707-19. A total AALS score (sum of the scores from all 5 subscales) may be utilized to assess ALS disease progression. In some embodiments, the rate of change in AALS scores for a subject can be determined from measuring the slope of total AALS scores. A slope of total AALS scores can be determined from scores collected at least two time-points of disease by the least squares method to obtain a points per month (points / month) change in total score. In certain embodiments, a long-term ALS survivor is an ALS subject that exhibits an increase in AALS score of less than about 1.5 to less than about 0.2 points / month, e.g., less than about 1.5 point / month, less than about 1 point / month, less than about 0.8 point / month, less than about 0.6 point / month, less than about 0.4 point / month, or less than about 0.2 point / month. In certain embodiments, a reference range for 4-HNE correlates with such an AALS rate of decline as described hereinabove. In certain embodiments, a subject having or suspected of having ALS is diagnosed a long-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject contains a level of 4-HNE equal to or lower than such a reference range for 4-HNE.

[0110] In certain embodiments, the reference range for 4-HNE ranges from about 0.5 µg / mL to about 15 µg / mL, from about 0.5 µg / mL to about 14 µg / mL, from about 0.5 µg / mL to about 13 µg / mL, from about 0.5 µg / mL to about 12 µg / mL, from about 0.5 µg / mL to about 11 µg / mL, or from about 0.5 µg / mL to about 10 µg / mL. In particular embodiments, the reference range for 4- HNE ranges from about 0.5 µg / mL to about 12 µg / mL. In certain embodiments, the reference range is about 0.5 µg / mL, about 1 µg / mL, about 2 µg / mL, about 3 µg / mL, about 4 µg / mL, about 5 µg / mL, about 6 µg / mL, about 7 µg / mL, about 8 µg / mL, about 9 µg / mL, about 10 µg / mL, 11 µg / mL, about 12 µg / mL, about 13 µg / mL or about 14 µg / mL, 15 µg / mL.

[0111] A long-term ALS survivor may, for example, be an ALS subject that survives longer NAI-1540949621v1 30Atty. Docket No.14678-018-228 than the mean ALS survival time. In certain embodiments, ALS mean survival time is about 3 years, about 4 years, about 5 years, about 2-4 years, or about 3-5 years. In certain embodiments, mean survival time is measured from the time of onset of ALS symptoms. In certain embodiments, mean survival time is measured from the time of diagnosis of ALS symptoms. In certain embodiments, a reference range for 4-HNE correlates with mean survival time as described hereinabove. In certain embodiments, a subject having or suspected of having ALS is diagnosed a long-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject contains a level of 4-HNE equal to or lower than such a reference range for 4-HNE.

[0112] In certain embodiments, the reference range for 4-HNE ranges from about 0.5 µg / mL to about 15 µg / mL, from about 0.5 µg / mL to about 14 µg / mL, from about 0.5 µg / mL to about 13 µg / mL, from about 0.5 µg / mL to about 12 µg / mL, from about 0.5 µg / mL to about 11 µg / mL, or from about 0.5 µg / mL to about 10 µg / mL. In particular embodiments, the reference range for 4- HNE ranges from about 0.5 µg / mL to about 12 µg / mL. In certain embodiments, the reference range is about 0.5 µg / mL, about 1 µg / mL, about 2 µg / mL, about 3 µg / mL, about 4 µg / mL, about 5 µg / mL, about 6 µg / mL, about 7 µg / mL, about 8 µg / mL, about 9 µg / mL, about 10 µg / mL, 11 µg / mL, about 12 µg / mL, about 13 µg / mL or about 14 µg / mL, 15 µg / mL.

[0113] In some embodiments, a subject is diagnosed as a long-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject is equal to or lower than a reference range of 4-HNE. In particular embodiments, such a diagnosis predicts that the subject have about a 30%, about a 40%, about a 50%, about a 60%, about a 70%, or about a 75% or greater chance of being a long-term ALS survivor.

[0114] In some embodiments, a subject is diagnosed as a long-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject is equal to or lower than a reference range of 4-HNE. In particular embodiments, the level of 4-HNE in the subject sample is about 0.5% to about 50%, about 0.5% to about 60%, about 0.5% to about 70%, about 0.5% to about 80%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 2% to about 50%, about 2% to about 60%, about 2% to about 70%, about 2% to about 80%, about 3% to about 50%, about 3% to about 60%, about 3% to about 70%, about 3% to about 80%, about 4% to about 50%, about 4% to about 60%, about 4% to about 70%, about 4% to about 80%, about 5% to about 50%, about 5% to about 60%, about NAI-1540949621v1 31Atty. Docket No.14678-018-228 5% to about 70%, or about 5% to about 80% lower than a reference range for 4-HNE. In some embodiments, the level of 4-HNE in the subject sample is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 15%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95% lower than a reference range for 4-HNE. In some embodiments, the level of 4-HNE in the subject sample is about 0.5-fold to about 10-fold, about 1-fold to about 9-fold, about 2-fold to about 8-fold, or about 3-fold to about 5-fold lower than a reference range for 4-HNE. In some embodiments, the level of 4-HNE in the subject sample is about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, or about more than 10-fold lower than a reference range for 4-HNE. Short-Term ALS Survivors

[0115] In certain aspects, presented herein are methods for predicting survival in a subject having or suspected of having ALS comprising (a) determining a level of 4-HNE in biological sample from a subject, for example, in a plasma or serum sample from the subject, and (b) diagnosing the subject as a short-term ALS survivor if the level of 4-HNE in the sample is greater than a reference range for 4-HNE. In certain embodiments, presented herein are methods for predicting survival in a subject having or suspected of having ALS comprising (a) obtaining or having obtained a biological sample from the subject, (b) determining a level of 4-HNE in biological sample from a subject, for example, in a plasma or serum sample from the subject, and (c) diagnosing the subject as a short-term ALS survivor if the level of 4-HNE in the sample is greater than a reference range for 4-HNE. In particular embodiments, a method of predicting survival in a subject having or suspected of having ALS may further comprise (d) administering (ii) a second therapeutic regimen to the subject if the subject is a short-term survivor.

[0116] A short-term ALS survivor may, for example, be an ALS subject that exhibits a particular ALSFRS-R score rate of decline. In certain embodiments, a short-term ALS survivor is an ALS subject that exhibits a decline in ALSFRS-R score greater than about 1 to about 3 NAI-1540949621v1 32Atty. Docket No.14678-018-228 points / month, e.g., greater than about 1 point / month, greater than about 1.2 points / month, greater than about 1.4 points / month, greater than about 1.6 points / month, greater than about 1.8 points / month, greater than about 2 points / month, greater than about 2.2 points / month, greater than about 2.4 points / month, greater than about 2.6 points / month, greater than about 2.8 points / month, or greater than about 3.0 points / month. In certain embodiments, a reference range for 4-HNE correlates with such an ALSFRS-R rate of decline as described hereinabove. In certain embodiments, a subject having or suspected of having ALS is diagnosed a short-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject contains a level of 4-HNE greater than such a reference range for 4-HNE.

[0117] In certain embodiments, the reference range for 4-HNE ranges from about 0.5 µg / mL to about 15 µg / mL, from about 0.5 µg / mL to about 14 µg / mL, from about 0.5 µg / mL to about 13 µg / mL, from about 0.5 µg / mL to about 12 µg / mL, from about 0.5 µg / mL to about 11 µg / mL, or from about 0.5 µg / mL to about 10 µg / mL. In particular embodiments, the reference range for 4- HNE ranges from about 0.5 µg / mL to about 12 µg / mL. In certain embodiments, the reference range is about 0.5 µg / mL, about 1 µg / mL, about 2 µg / mL, about 3 µg / mL, about 4 µg / mL, about 5 µg / mL, about 6 µg / mL, about 7 µg / mL, about 8 µg / mL, about 9 µg / mL, about 10 µg / mL, 11 µg / mL, about 12 µg / mL, about 13 µg / mL or about 14 µg / mL, 15 µg / mL.

[0118] A short-term ALS survivor may, for example, be an ALS subject that exhibits a particular AALS score rate of decline. In certain embodiments, a short-term ALS survivor is an ALS subject that exhibits an increase in AALS score greater than about 1.5 points / month to about 4 points / month, e.g., more than about 4 points / month, more than about 3.5 points / month, more than about 3 points / month, more than about 2.5 points / month, more than about 2 points / month, or more than about 1.5 points / month. In certain embodiments, a reference range for 4-HNE correlates with such an AALS rate of decline as described hereinabove. In certain embodiments, a subject having or suspected of having ALS is diagnosed a short-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject contains a level of 4-HNE greater than such a reference range for 4-HNE.

[0119] In certain embodiments, the reference range for 4-HNE ranges from about 0.5 µg / mL to about 15 µg / mL, from about 0.5 µg / mL to about 14 µg / mL, from about 0.5 µg / mL to about 13 µg / mL, from about 0.5 µg / mL to about 12 µg / mL, from about 0.5 µg / mL to about 11 µg / mL, or from about 0.5 µg / mL to about 10 µg / mL. In particular embodiments, the reference range for 4- NAI-1540949621v1 33Atty. Docket No.14678-018-228 HNE ranges from about 0.5 µg / mL to about 12 µg / mL. In certain embodiments, the reference range is about 0.5 µg / mL, about 1 µg / mL, about 2 µg / mL, about 3 µg / mL, about 4 µg / mL, about 5 µg / mL, about 6 µg / mL, about 7 µg / mL, about 8 µg / mL, about 9 µg / mL, about 10 µg / mL, 11 µg / mL, about 12 µg / mL, about 13 µg / mL or about 14 µg / mL, 15 µg / mL.

[0120] A short-term ALS survivor may, for example, be an ALS subject that survives less than the mean ALS survival time. In certain embodiments, ALS mean survival time is about 3 years, about 4 years, about 5 years, about 2-4 years, or about 3-5 years. In certain embodiments, a reference range for 4-HNE correlates with mean survival time as described hereinabove. In certain embodiments, a subject having or suspected of having ALS is diagnosed a short-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject contains a level of 4-HNE greater than such a reference range for 4-HNE.

[0121] In certain embodiments, the reference range for 4-HNE ranges from about 0.5 µg / mL to about 15 µg / mL, from about 0.5 µg / mL to about 14 µg / mL, from about 0.5 µg / mL to about 13 µg / mL, from about 0.5 µg / mL to about 12 µg / mL, from about 0.5 µg / mL to about 11 µg / mL, or from about 0.5 µg / mL to about 10 µg / mL. In particular embodiments, the reference range for 4- HNE ranges from about 0.5 µg / mL to about 12 µg / mL. In certain embodiments, the reference range is about 0.5 µg / mL, about 1 µg / mL, about 2 µg / mL, about 3 µg / mL, about 4 µg / mL, about 5 µg / mL, about 6 µg / mL, about 7 µg / mL, about 8 µg / mL, about 9 µg / mL, about 10 µg / mL, 11 µg / mL, about 12 µg / mL, about 13 µg / mL or about 14 µg / mL, 15 µg / mL.

[0122] In some embodiments, a subject is diagnosed as a short-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject is greater than a reference range of 4-HNE. In particular embodiments, such a diagnosis predicts that the subject have about a 30%, about a 40%, about a 50%, about a 60%, about a 70%, or about a 75% or greater chance of being a short-term ALS survivor.

[0123] In some embodiments, a subject is diagnosed as a short-term ALS survivor if the level of 4-HNE in a sample (e.g., a serum or plasma sample) from the subject is greater than a reference range of 4-HNE. In particular embodiments, the level of 4-HNE in the subject sample is about 0.5% to about 50%, about 0.5% to about 60%, about 0.5% to about 70%, about 0.5% to about 80%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 2% to about 50%, about 2% to about 60%, about 2% to about 70%, about 2% to about 80%, about 3% to about 50%, about 3% to about 60%, about 3% to about 70%, NAI-1540949621v1 34Atty. Docket No.14678-018-228 about 3% to about 80%, about 4% to about 50%, about 4% to about 60%, about 4% to about 70%, about 4% to about 80%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, or about 5% to about 80% greater than a reference range for 4-HNE. In some embodiments, the level of 4-HNE in the subject sample is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 15%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95% greater than a reference range for 4-HNE. In some embodiments, the level of 4-HNE in the subject sample is about 0.5-fold to about 10-fold, about 1-fold to about 9-fold, about 2-fold to about 8-fold, or about 3-fold to about 5-fold greater than a reference range for 4-HNE. In some embodiments, the level of 4-HNE in the subject sample is about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, or about more than 10-fold greater than a reference range for 4-HNE. 7.2 4-HNE

[0124] 4-hydroxynonenal (4-HNE; CAS Reg. No. 75899-68-2). 4-HNE (also known as 4- hydroxy-2-nonenal) is represented in formula I: I.

[0125] 4-HNE is afatty acids and, without being bound by any particular theory, is frequently measured as an indicator of lipid peroxidation and oxidative stress. Methods of determining a level of 4-HNE in a biological sample are well known in the art. Non-limiting examples of biological samples include cerebrospinal fluid (CSF), serum, blood, plasma, peripheral blood mononuclear cells (PBMCs), urine, saliva, tissue, and the like. In certain embodiments, the biological sample is a serum or plasma sample. A biological sample can be collected from a patient using standard techniques known in the art. In some embodiments, a biological sample taken from a patient can be a whole NAI-1540949621v1 35Atty. Docket No.14678-018-228 blood collection.

[0126] In certain embodiments, a level of 4-HNE in a sample (e.g., serum or plasma) can be determined using any suitable method for 4-HNE determination known in the art. Such methods include, but are not limited to, immunoassays (e.g., enzyme linked-immunosorbent assay (ELISA)), protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, protein immunostaining, electrophoresis analysis, competitive binding assays, functional protein assays, protein microarray, chromatography or spectrometry methods (e.g., high-performance liquid chromatography (HPLC), mass spectrometry, liquid chromatography-mass spectrometry (LC / MS), capillary electrophoresis (CE)-MS, or any separating front end coupled with MS detection and quantification), cytometric bead array analysis, immunonephelometric assays, fluorescence activated cell sorting (FACS) analysis, microscopic analysis, microarray, and the like.

[0127] For example, a number of immunoassays to measure 4-HNE levels in serum and plasma are commercially available. In addition, since 4-HNE displays a relatively fast half-life of less than 2 minutes in normal physiological conditions, assays can detect levels of protein adducts of 4-HNE as a surrogate for 4-HNE. ELISA kits are commercially available for protein adducts of 4-HNE. An assay for determining 4-HNE adduct levels in serum and plasma is described, for instance, in Monroe et al., Bio Protoc (2019) 9(19):e3383. An exemplary assay for determining 4-HNE levels in serum samples from a subject is described in Section 8.

[0128] In some embodiments, the 4-HNE present in a biological sample at low levels may be determined using ultrasensitive methodologies and devices specifically designed for detecting low amounts of 4-HNE in a sample. Examples of such methodologies and devices include but are not limited to microfluidic analytical systems (see, e.g., Martel JM and Toner M., Annu Rev Biomed Eng (2014) Jul.11; 16:371-96); ultra-sensitive ELISA assays (see, e.g., Schubert S et al., Sci Rep (2015) 5, 11034); and nanoparticle-based systems (see, e.g., Li F et al., Biosensors and Bioelectronics (2015) 68, 626-632). One of skill in the art would appreciate that the method of determining the level of 4-HNE in a biological sample (e.g., serum or plasma) may be chosen on the basis of the type of sample. In specific embodiments, the 4-HNE level may be determined in a biological sample (e.g., serum, plasma) using an ELISA. Kits for use in determining a level of 4-HNE are disclosed herein and are further described in Section 7.6.

[0129] In certain embodiments of the methods provided herein, a level of 4-HNE can be NAI-1540949621v1 36Atty. Docket No.14678-018-228 determined in a biological sample (e.g., serum, plasma) collected from a subject who is suspected of having ALS. In certain embodiments of the methods provided herein, a level of 4- HNE can be determined in a biological sample (e.g., serum, plasma) collected from a subject who is genetically predisposed to having ALS. In some embodiments, a level of 4-HNE can be determined in a biological sample (e.g., serum, plasma) collected from a subject on the date of disease onset. In some embodiments, a level of 4-HNE can be determined in a biological sample (e.g., serum, plasma) collected from a subject about 1 day, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, or more than about 5 years from the date of disease onset. In some embodiments, a level of 4-HNE can be determined in a biological sample (e.g., serum, plasma) collected from a subject on the date of disease diagnosis. In some embodiments, a level of 4-HNE can be determined in a biological sample (e.g., serum, plasma) collected from a subject about 1 day, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, or more than about 5 years from the date of disease diagnosis. In some embodiments, a level of 4-HNE can be determined in more than one biological sample (e.g., serum, plasma) collected from a subject. In some embodiments, a level of 4-HNE can be determined in about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, or more than about 200 biological samples (e.g., serum, plasma) collected from a single subject. In some embodiments, more than one biological sample (e.g., serum, plasma) can be collected from a subject in a single day. In some embodiments, more than one biological sample (e.g., serum, plasma) can be collected from a subject at regular time intervals between collection of samples. In some embodiments, more than one biological sample (e.g., serum, plasma) can be collected from a subject every day, every 2 days, every 3 days, every 4 days, every 5 days, every NAI-1540949621v1 37Atty. Docket No.14678-018-228 6 days, every week, every other week, or every month. In some embodiments, more than one biological sample (e.g., serum, plasma) can be collected from a subject at irregular intervals between collection of samples. In some embodiments, a mean level of 4-HNE can be determined from the more than one biological sample (e.g., serum, plasma) collected from a single subject. In some embodiments, a level of 4-HNE can be determined in at least one biological sample collected on the date of disease onset and in another at least one biological sample collected on a date after disease onset. In some embodiments, a level of 4-HNE can be determined in at least one biological sample collected on the date of ALS diagnosis and in another at least one biological sample collected on a date after diagnosis of ALS. In some embodiments, a level of 4- HNE can be determined in at least one biological sample collected on the date of disease onset and in another at least one biological sample collected on the date of ALS diagnosis. In some embodiments, a level of 4-HNE can be determined in at least one biological sample collected on the date of disease onset and in another at least one biological sample collected before ALS- related death. In some embodiments, a level of 4-HNE can be determined in at least one biological sample collected on the date of ALS disease diagnosis and in another at least one biological sample collected before ALS-related death. In some embodiments, at least one biological sample for use in the methods disclosed herein can be collected at least about one day, at least about 1 week, least about 1 month, least about 2 months, at least about 4 months, or at least about 6 months before ALS-related death.

[0130] In certain embodiments of the methods provided herein, a level of 4-HNE can be determined in successive biological samples (e.g., serum, plasma) collected from a subject who is suspected of or having ALS. It will be understood that “successive biological samples” means samples taken from the ALS patient on different days, e.g., on different visits to a hospital, clinician’s office or wherever blood samples are taken from the ALS patient. For example, “at least one successive biological sample” refers to a sample taken on a day following the day on which in initial or first sample was taken from the ALS patient. In certain embodiments, successive biological samples comprise at least two samples. In certain embodiments, successive biological samples comprise at least three samples. In certain embodiments, successive biological samples comprise at least four samples. In certain embodiments, successive biological samples comprise at least five samples. In certain embodiments, successive biological samples comprise at least six samples. In certain embodiments, successive biological NAI-1540949621v1 38Atty. Docket No.14678-018-228 samples comprise at least seven samples. In some embodiments, the successive biological samples are consecutive. In some embodiments, the successive biological samples are non- consecutive.

[0131] In certain embodiments, a 4-HNE reference range as described herein is obtained from one or more healthy individuals. In certain embodiments, a 4-HNE reference range is a mean average level from healthy individuals. In some embodiments, a 4-HNE reference range is one standard deviation above the mean average level from a group of healthy individuals. In some embodiments, a 4-HNE reference range is within a range of 4-HNE levels encompassing a mean average level from a group of healthy individuals.

[0132] The number of healthy individuals that contribute to a 4-HNE reference range can, for example, be at least 3. In some embodiments, the number of healthy individuals that contribute to a 4-HNE reference range can, for example, be at least 10. In certain embodiments, the number of healthy individuals is between about 3 to about 10,000. In other embodiments, the number of healthy individuals is between about 5 to about 1,000, or between about 8 to about 40. In some embodiments, the number healthy individuals contributing to the 4-HNE reference range is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, at least 1,000, at least 2,000, or at least 3,000.

[0133] In certain embodiments, the 4-HNE reference range is from healthy individuals that are age-matched to the ALS patient. In certain embodiments, the 4-HNE reference range is from healthy individuals that are gender-matched to the ALS patient. In certain embodiments, the 4- HNE reference range is from healthy individuals that share the same ethnicity as the ALS patient. In certain embodiments, the 4-HNE reference range is an average level of 4-HNE measured in serum or plasma samples collected from at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more than 10 healthy subjects having the same age, gender, and / or ethnicity as the patient. 7.3 Methods of ALS Treatment

[0134] In certain embodiments, methods disclosed herein treat an ALS patient based on prediction of survival time for the ALS subject (e.g., short-term or long-term survivor). In certain embodiments, methods disclosed herein treat an ALS patient based on prediction of ALS NAI-1540949621v1 39Atty. Docket No.14678-018-228 disease progression (e.g., rapid or slow progression) to disease endpoints, e.g., death or a need for implementation of invasive respiratory measures. Any of the ALS therapies disclosed in Section 7.4 can be used in the methods described herein.

[0135] In certain embodiments, methods of the present disclosure are directed, in part, toward treating ALS in a subject suspected of having or diagnosed with ALS. As used herein and unless otherwise indicated, the terms “treat,” “treating,” “treatment,” and “ameliorating” are used interchangeably herein, and mean an alleviation, in whole or in part, of a disorder, disease or condition, such as ALS, or one or more of the symptoms associated with a disorder, disease, or condition, such as ALS, or slowing or halting of further progression or worsening of those symptoms, or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself, such as ALS. Non-limiting examples of symptoms of ALS include dyspnea, orthopnea, muscle weakness, dysphagia, and dysarthria. A therapeutically effective amount of an ALS therapy is an amount that treats a subject having or diagnosed with ALS as “treats” is used herein.

[0136] In certain embodiments, provided herein is a method for treating ALS in a patient diagnosed therewith, the method comprising determining whether a level of 4-HNE in a sample, e.g., a serum or plasma sample, from a diagnosed with or suspected of having ALS is at or below a reference range and administering to the patient a first therapeutic regimen if the subject is predicted to be a long-term survivor, or a second therapeutic regimen if the subject is predicted to be a short-term survivor. Methods of predicting a subject to be a long-term survivor or a short-term survivor by determining and comparing the 4-HNE expression in a biological sample to a reference 4-HNE range are disclosed herein (see Section 7.1). As used herein, a “therapeutic regimen” may refer to an ALS therapy. For example, a therapeutic regimen may refer to a dosage of one or more ALS therapies

[0137] In certain embodiments, a first therapeutic regimen administered to a subject predicted to be a long-term survivor according to the methods herein can be the same ALS therapy as a second therapeutic regimen administered to a subject predicted to be a short-term survivor according to the methods herein. In some embodiments, the second therapeutic regimen administered to the subject predicted to be a short-term survivor comprises a higher dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen to the subject predicted to be a long-term survivor. In some embodiments, the second therapeutic regimen administered to the subject predicted to be a short-term survivor comprises NAI-1540949621v1 40Atty. Docket No.14678-018-228 at least about a 0.5-fold to 50-fold, about a 1-fold to 25-fold, or about a 1-fold to 10-fold higher dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen to the subject predicted to be a long-term survivor. In some embodiments, the second therapeutic regimen administered to the subject predicted to be a short-term survivor comprises a lower dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen to a subject predicted to be a long-term survivor. In some embodiments, the second therapeutic regimen administered to the subject predicted to be a short-term survivor comprises at least about a 0.5-fold to 50-fold, about a 1-fold to 25-fold, or about a 1-fold to 10-fold lower dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen to a subject predicted to be a long-term survivor.

[0138] In some embodiments, the second therapeutic regimen is administered to the subject predicted to be a short-term survivor with increased frequency compared to the frequency of the ALS therapy administered in the first therapeutic regimen to a subject predicted to be a long- term survivor. In some embodiments, the second therapeutic regimen is administered to the subject predicted to be a short-term survivor with increased frequency of at least about 1 more to about 10 more, about 2 more to about 10 more, or about 3 more to about 10 more administrations compared to the frequency of the ALS therapy administered in the first therapeutic regimen to a subject predicted to be a long-term survivor.

[0139] In some embodiments, the second therapeutic regimen is administered to the subject predicted to be a short-term survivor with decreased frequency compared to the frequency of the ALS therapy administered in the first therapeutic regimen to a subject predicted to be a long- term survivor. In some embodiments, the second therapeutic regimen is administered to the subject predicted to be a short-term survivor with decreased frequency of about 1 less to about 10 less, about 2 less to about 10 less, or about 3 less to about 10 less administrations compared to the frequency of the ALS therapy administered in the first therapeutic regimen to a subject predicted to be a long-term survivor.

[0140] In certain embodiments, a first therapeutic regimen administered to a subject predicted to be a long-term survivor according to the methods herein can be a different ALS therapy than a second therapeutic regimen administered to a subject predicted to be a short-term survivor according to the methods herein. NAI-1540949621v1 41Atty. Docket No.14678-018-228

[0141] In some embodiments, a first therapeutic regimen can be administered to a subject predicted to be a long-term survivor one or more times. In some embodiments, a first therapeutic regimen can be administered to a subject predicted to be a long-term survivor at least about 1 to 10, about 1 to 9, about 1 to 8, about 1 to 7, about 1 to 6, about 1 to 5, about 1 to 4, about 1 to 3, or about 1 to 2 more times after the first administration. In some embodiments, a biological sample can be collected from the subject to measure 4-HNE levels according to the methods disclosed herein before administration of any additional first therapeutic regimen administration.

[0142] In some embodiments, a second therapeutic regimen can be administered to a subject predicted to be a short-term survivor one or more times. In some embodiments, a second therapeutic regimen can be administered to a subject predicted to be a short-term survivor at least about 1 to 10, about 1 to 9, about 1 to 8, about 1 to 7, about 1 to 6, about 1 to 5, about 1 to 4, about 1 to 3, or about 1 to 2 more times after the first administration. In some embodiments, a biological sample can be collected from the subject to measure 4-HNE levels according to the methods disclosed herein before administration of any additional second therapeutic regimen administration.

[0143] In certain embodiments, methods disclosed herein comprise administering to a subject predicted to be a short-term ALS survivor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a short-term ALS survivor in that serum or plasma collected from the subject comprises a 4-HNE level greater than a 4-HNE reference range. In certain embodiments, methods disclosed herein comprise administering to a subject predicted to be a rapid ALS progressor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a rapid ALS progressor in that serum or plasma collected from the subject comprises a 4-HNE level greater than a 4-HNE reference range.

[0144] In certain embodiments, methods disclosed herein comprise administering to a subject predicted to be a long-term ALS survivor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a long-term ALS survivor in that serum or plasma collected from the subject comprises a 4-HNE level equal to or lower than a 4-HNE reference range. In certain embodiments, methods disclosed herein comprise administering to a subject predicted to be a slow ALS progressor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a slow ALS progressor in that serum or plasma collected from the subject comprises a 4-HNE level equal to or lower than a 4-HNE reference range. NAI-1540949621v1 42Atty. Docket No.14678-018-228

[0145] In certain embodiments, methods provided herein comprise determining the impact of an ALS therapy on survival of a subject having or suspected of having ALS. In some embodiments, the method comprises (a) determining a level of 4-HNE in a plasma or serum sample from the subject; (b) diagnosing the subject as: (i) a long-term ALS survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short- term ALS survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE; (c) administering an ALS therapy to the subject; (d) determining a level of 4-HNE in a plasma or serum sample from the subject following (c); and (e) comparing the level of 4-HNE in (a) to the 4-HNE level in (d), thereby determining the impact of the ALS therapy on survival of the subject.

[0146] In some embodiments, the ALS therapy improves survival of the subject if the difference in the level of 4-HNE determined in a plasma or serum sample from the subject before administering the ALS therapy is equal to or lower than the level of 4-HNE determined in a plasma or serum sample from the subject collected after administering the ALS therapy. In some embodiments, the ALS therapy improves survival of the subject if the difference in the level of 4-HNE determined before administering the ALS therapy is equal to or lower than the level of 4- HNE determined after administering the ALS therapy and the level of 4-HNE determined after administering the ALS therapy is equal to or lower than a 4-HNE reference range.

[0147] In some embodiments, the ALS therapy improves survival of the subject compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy. In some embodiments, the ALS therapy improves survival of the subject at least about 0.2-fold to about 50-fold, at least about 0.5-fold to about 50-fold, at least about 1-fold to about 40-fold, at least about 1.5-fold to about 30-fold, or at least about 2- fold to about 20-fold compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy. In some embodiments, the ALS therapy improves survival of the subject at least about 0.2-fold, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, or about 50-fold NAI-1540949621v1 43Atty. Docket No.14678-018-228 compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy.

[0148] In some embodiments, the ALS therapy improves survival of the subject at least about 1% to 99%, about 1% to 90%, about 1% to 80%, about 1% to 85%, about 1% to 80%, about 1% to 75%, about 1% to 70%, about 1% to 65%, about 1% to 60%, about 1% to 65%, about 1% to 50%, about 1% to 55%, about 1% to 40%, about 1% to 45%, about 1% to 30%, about 1% to 25%, about 1% to 20%, about 1% to 15%, about 1% to 10%, or about 1% to 5% compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy.

[0149] In some embodiments, the ALS therapy improves survival of the subject at least about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy.

[0150] In some embodiments, the ALS therapy improves survival of the subject by about 1 day to about 10 years, about 1 week to about 9 years, about 2 weeks to about 8 years, about 3 weeks to about 7 years, about 1 month to about 6 years, or about 2 months to about 8 years compared to the survival time predicted according to the methods disclosed herein of the subject before administration of the ALS therapy.

[0151] In some embodiments, the ALS therapy improves survival of the subject by about 1 day, about 3 days, about 5 days, about 1 week, about 1.5 weeks, about 2 weeks, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more than about 10 years compared to the survival time predicted according to the methods disclosed herein of the subject NAI-1540949621v1 44Atty. Docket No.14678-018-228 before administration of the ALS therapy.

[0152] In some embodiments, the ALS therapy worsens survival of the subject if the difference in the level of 4-HNE in a plasma or serum sample from the subject determined before administering the ALS therapy is higher than the level of 4-HNE determined in a plasma or serum sample from the subject collected after administering the ALS therapy. In some embodiments, the ALS therapy worsens survival of the subject if the difference in the level of 4- HNE determined before administering the ALS therapy is higher than the level of 4-HNE determined after administering the ALS therapy and the level of 4-HNE determined after administering the ALS therapy is greater than a 4-HNE reference range.

[0153] In some embodiments, the ALS therapy worsens survival of the subject compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy. In some embodiments, the ALS therapy worsens survival of the subject at least about 0.5-fold to about 50-fold, at least about 1-fold to about 40-fold, at least about 1.5-fold to about 30-fold, or at least about 2-fold to about 20-fold compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy. In some embodiments, the ALS therapy worsens survival of the subject at least about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, or about 50-fold compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy.

[0154] In some embodiments, the ALS therapy worsens survival of the subject at least about 1% to 99%, about 1% to 90%, about 1% to 80%, about 1% to 85%, about 1% to 80%, about 1% to 75%, about 1% to 70%, about 1% to 65%, about 1% to 60%, about 1% to 65%, about 1% to 50%, about 1% to 55%, about 1% to 40%, about 1% to 45%, about 1% to 30%, about 1% to 25%, about 1% to 20%, about 1% to 15%, about 1% to 10%, or about 1% to 5% compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy. In some embodiments, the ALS therapy worsens survival of the subject at least about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about NAI-1540949621v1 45Atty. Docket No.14678-018-228 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% compared to the survival predicted according to the methods disclosed herein of the subject before administration of the ALS therapy.

[0155] In some embodiments, the ALS therapy decreases survival of the subject by about 1 day to about 10 years, about 1 week to about 9 years, about 2 weeks to about 8 years, about 3 weeks to about 7 years, about 1 month to about 6 years, or about 2 months to about 8 years compared to the survival time predicted according to the methods disclosed herein of the subject before administration of the ALS therapy. In some embodiments, the ALS therapy decreases survival of the subject by about 1 day, about 3 days, about 5 days, about 1 week, about 1.5 weeks, about 2 weeks, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more than about 10 years compared to the survival time predicted according to the methods disclosed herein of the subject before administration of the ALS therapy.

[0156] In certain embodiments, methods herein can comprise enrolling a patient in a clinical trial to test an ALS therapy if the 4-HNE level in a serum sample is the same or less than a 4- HNE reference range. In certain embodiments, the method comprises enrolling a patient in a clinical trial to test an ALS therapy if the 4-HNE level in a serum sample is greater than a 4-HNE reference range. In certain embodiments, the method further comprises administering an ALS therapy to the enrolled patient.

[0157] In certain embodiments, methods disclosed herein provide a method of predicting clinical benefit of an ALS therapy. In some embodiments, methods of predicting the clinical benefit of an ALS therapy can comprise: (a) administering the ALS therapy to a subject; (b) collecting a plasma or serum sample from the subject after administering the ALS therapy; (c) determining the level of 4-HNE in the sample collected from the subject, wherein the level of 4- NAI-1540949621v1 46Atty. Docket No.14678-018-228 HNE in the sample acts as a surrogate endpoint; (d) predicting the clinical benefit of the ALS therapy. As used herein an “endpoint” refers to a targeted outcome of a clinical trial that is analyzed to determine the efficacy and safety of the therapy being studied. A “surrogate endpoint” is understood to refer to a measurable marker that replaces traditional ALS study endpoints (e.g., death, invasive ventilation) to assess the outcome of a clinical trial. In some embodiments, a 4-HNE level in a biological sample collected from a patient can be a surrogate endpoint. In some embodiments, methods of predicting the clinical benefit of an ALS therapy can predict a favorable clinical benefit if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE. In some embodiments, a favorable clinical benefit of the ALS therapy as determined according to the methods disclosed herein is correlated with slow disease progression. In some embodiments, a favorable clinical benefit of the ALS therapy as determined according to the methods disclosed herein is correlated with increased survival time. In some embodiments, methods of predicting the clinical benefit of an ALS therapy can predict an unfavorable clinical benefit if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE. In some embodiments, an unfavorable clinical benefit of the ALS therapy as determined according to the methods disclosed herein is correlated with fast disease progression. In some embodiments, an unfavorable clinical benefit of the ALS therapy as determined according to the methods disclosed herein is correlated with decreased survival time. In some embodiments, use of the level of 4-HNE in the sample as surrogate endpoint can predict clinical benefit in a clinical trial of at least one investigational therapy for ALS. 7.4 ALS Therapies

[0158] As further disclosed in Section 7.3, any one or more of the ALS therapies disclosed herein can comprise a therapeutic regimen for treating ALS. For the methods provided herein such as, for instance, methods for predicting survival time in a subject diagnosed as having ALS or suspected of having ALS methods and / or methods for predicting progression of ALS in a subject, a An ALS therapy for use in the disclosed methods can be any therapy that is administered to an ALS patient to treat ALS. In certain embodiments, provided herein are methods of administering one or more agents that treats ALS (e.g., based on the agent’s product label or known to one skilled in the art e.g., Mead RJ et al., Nat Rev Drug Discov.2022 Dec 21:1–28). In certain embodiments, an agent that treats ALS can be an immunotherapy, a cell- based therapy, or a combination thereof. In some embodiments, the ALS therapy is a Treg NAI-1540949621v1 47Atty. Docket No.14678-018-228 therapy (Section 7.4.1). In some embodiments, the ALS therapy is an interleukin-2 (“IL-2”) therapy (Section 7.4.2). In some embodiments, the ALS therapy is an IL-2 / abatacept combination therapy (Section 7.4.3).

[0159] In certain embodiments, methods provided herein can comprise administering the ALS therapy to the ALS patient to treat ALS, e.g., to mitigate a symptom of ALS. In certain embodiments, methods provided herein can comprise administering the ALS therapy to the ALS patient in a clinical trial to test the ALS therapy.

[0160] In certain embodiments, an agent to be used according to the methods disclosed herein can be an FDA approved drug indicated for the treatment of ALS. Non-limiting examples of such an agent can include: Relyvrio™ (AMX0035; a 3:1 combination of sodium phenylbutyrate (4-phenyl butyric acid sodium salt) and taurursodiol (2-[(3α, 7β-dihydroxy-24- oxo-5β-cholan-24-yl) amino] ethane sulfonic acid, dihydrate) also known as tauroursodeoxycholic acid or TUDCA); edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one]; and / or riluzole (2-amino-6-(trifluoromethoxy)benzothiazole).

[0161] In certain embodiments, non-limiting examples of an agent that can be used according to the methods disclosed herein for the treatment of ALS can include Tocilizumab, ACTH / Acthar gel, IL-2, Ibudilast, Pentoxifylline, Masitinib, NP001, Thalidomide, Glatiramer Acetate, CC100, Fingolimod, Minocycline, Pioglitazone, DNL747, Fasudil, Ono-2506, Anakinra, RNS60, Retigabine, Memantine, Talampanel, Perampanel, Mexiletine, Ceftriaxone, Celecoxib, Lamotrigine, Nimodipine, Gabapentin, Epi-589, N-Acetylcysteine, Vitamine E, Coenzyme Q10, Selegiline, Rasagiline, Cu(II)-ATSM, BIIB067, Pyrimethamine, Levosimendan, Tirasemtiv, CK-2127107IGF-1, Xaliproden, EPO, BDNF, GM604, CNTF, SB509, VM202, sNN0029, KP-100, Dexpramipexole, Olexosime, Creatine, Acetyl-L-carnitine, Lithium, Rapamycin, Tamoxifen, Ozanezumab, GDC-0134, YAM80, Methylcobalamin, Pimozide, Omigapil / TCH346, Deferiprone, Arimoclomol, EH301, BIIB078, and / or MD1003 (see e.g., Wobst HJ et al., Med Res Rev.2020 Jul;40(4):1352-1384).

[0162] In certain embodiments, an agent to be used according to the methods disclosed herein can be a drug for controlling one or more symptoms of ALS. Non-limiting examples of such agents can include topiramate (2,3:4,5-Bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate); gabapentin (2-[1-(aminomethyl)cyclohexyl]acetic acid); lamotrigine (6-(2,3- dichlorophenyl)-1,2,4-triazine-3,5-diamine); talampanel ((8R)-7-Acetyl-5-(4-aminophenyl)-8,9- NAI-1540949621v1 48Atty. Docket No.14678-018-228 dihydro-8-methyl-7H-1,3-dioxolo[4,5-h] [2,3]benzodiazepine); ceftriaxone ((6R,7R)-7-[[(2Z)-2- (2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetyl]amino]-3-[(2-methyl-5,6-dioxo-1H-1,2,4- triazin-3-yl)sulfanylmethyl]-8-ox o-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid); an inhibitor of glutamate carboxypeptidase II; dextromethorphan / quinidine; TUDCA (tauroursodeoxycholic acid); baclofen (4-amino-3-(4-chlorophenyl)butanoic acid); diazepam (7- chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2-one); trihexyphenidyl (1-cyclohexyl-1- phenyl-3-piperidin-1-ylpropan-1-ol); and / or amitriptyline (N,N-dimethyl-3-(2- tricyclo[9.4.0.03,8]pentadeca-1(15),3,5,7,11,13-hexaenylidene)propan-1-amine).

[0163] In some embodiments, the methods provided herein can comprise administering to the subject a drug that has been repurposed for the treatment of ALS (e.g., edaravone, rasagiline, AMX0035, masitinib, and / or acrimonclomol). See also Kiernan et al., Nat Rev Neurol.2021 Feb;17(2):104-118.

[0164] In certain embodiments, additional non-limiting examples of an agent to be used according to the methods disclosed herein can include abacavir, dolutegravir, lamivudine, curcumin, sodium phenylbutyrate, naltrexone, tauroursodeoxycholic acid (TUDCA), methylcobalamin, dextromethorphan hydrobromide, quinidine sulfate, nuedexta, ropinirol, metformin, tamoxifen, trimetazidine, masitinib, CuATSM (diacetylbis(N(4)- methylthiosemicarbazonato)copper(II)), and / or reldesemtiv. See also Pridmore W, (2021). RRNMF Neuromuscular Journal, 2(1), 36–40.

[0165] In certain embodiments, an ALS therapy can, for example, comprise TIGLUTIK (thickened riluzole), EXSERVAN™ (riluzole oral film), BHV-0223 (sublingual riluzole), NUEDEXTA® (dextromethorphan HBr and quinidine sulfate), ravulizumab-cwvz (ULTOMIRIS®), mesenchymal stem cell (MSC)-neurotrophic factor (NTF) cells (e.g., NUROWN®), MASITINIB (an oral tyrosine kinase inhibitor), TOFERSEN (BIIB067, IONIS- SOD1Rx, Ionis Pharmaceuticals and Biogen), APB-102 (Apic Bio), H.P. ACTHAR GEL (Mallinckrodt Pharmaceuticals), MN-166 (MediciNova), GM-6 (Genervon), GILENYA (fingolimod, ALS TDI), ARIMOCLOMOL (Orph-001,Orphazyme), NP001 (Neuraltus), VM202 (VM Biopharma), RELDESEMTIV (Cytokinetics), NUROWN (BrainStorm Cell Therapeutics), NSI-566 (Neuralstem), MEXILETINE, acamprosate, baclofen, cinacalcet, sulfisoxazole, torasemide, or any combination thereof.

[0166] In certain embodiments, an ALS therapy for use herein comprises administering a NAI-1540949621v1 49Atty. Docket No.14678-018-228 gene therapy to the patient. In some embodiments, a gene therapy for treating ALS according to the methods disclosed herein can be one that targets one or more gene mutations associated with ALS. In some embodiments, a gene therapy for treating ALS according to the methods disclosed herein can be one that targets one or more mutations in the genes SOD1, C9orf72, and / or FUS. 7.4.1 Treg Therapy

[0167] In certain embodiments of the methods provided herein, the ALS therapy is a T regulatory cell (“Treg”) therapy. In certain embodiments, the ALS therapy comprises a Treg infusion. In certain embodiments, the ALS therapy comprises a plurality of Treg infusions. Administering Treg therapy to ALS patients has been shown to slow progression rates of the disease, and Treg suppressive function in some ALS patients correlates with the slowing of disease progression (Thonhoff, J.R. et al., Neurology-Neuroimmunology Neuroinflammation 5(4):e465 (2018)). However, as demonstrated in the Examples below, not all ALS patients are responsive to Treg therapy. In some embodiments, methods are provided herein to stratify patients that will likely be, or are, responsive to Treg therapy (“responders”), and those that will likely not be, or are not, responsive to Treg therapy (“non-responders”).

[0168] Certain methods provided herein may be performed with any steps of isolating, expanding, and administering Treg therapy to subjects (e.g., ALS patients) that are known in the art or disclosed herein. Exemplary methods of producing obtaining, enriching for and ex-vivo expanding a population of Tregs are described in International PCT Application No. PCT / US2020 / 063378, which is incorporated by reference herein in its entirety, in particular for its teaching of such methods.

[0169] As a non-limiting example, in some embodiments, the ALS therapy comprises collecting white blood cells from the ALS patient (leukapheresis); isolating and expanding ex vivo Tregs from the collected white blood cells; and administering the expanded Tregs intravenously (infusion) to the ALS patient.

[0170] In some embodiments, about 1 × 106to about 2 × 106, about 2 × 106to about 3 × 106, about 3 × 106to about 4 × 106, about 4 × 106to about 5 × 106, about 5 × 106to about 6 × 106, about 6 × 106to about 7 × 106, about 7 × 106to about 8 × 106, about 8 × 106to about 9 × 106, about 9 × 106to about 1 × 107, about 1 × 107to about 2 × 107, about 2 × 107to about 3 × 107, about 3 × 107to about 4 × 107, about 4 × 107to about 5 × 107, about 5 × 107to about 6 × 107, about 6 × 107to about 7 × 107, about 7 × 107to about 8 × 107, about 8 × 107to about 9 × 107, NAI-1540949621v1 50Atty. Docket No.14678-018-228 about 9 × 107to about 1 × 108, about 1 × 108to about 2 × 108, about 2 × 108to about 3 × 108, about 3 × 108to about 4 × 108, about 4 × 108to about 5 × 108, about 5 × 108to about 6 × 108, about 6 × 108to about 7 × 108, about 7 × 108to about 8 × 108, about 8 × 108to about 9 × 108, about 9 × 108to about 1 × 109Tregs per kg of body weight of the subject are administered. In some embodiments, 1 x106Tregs (+ / - 10%) per kg of body weight of the subject are administered.

[0171] In some embodiments, about 1 × 106to about 2 × 106, about 2 × 106to about 3 × 106, about 3 × 106to about 4 × 106, about 4 × 106to about 5 × 106, about 5 × 106to about 6 × 106, about 6 × 106to about 7 × 106, about 7 × 106to about 8 × 106, about 8 × 106to about 9 × 106, about 9 × 106to about 1 × 107, about 1 × 107to about 2 × 107, about 2 × 107to about 3 × 107, about 3 × 107to about 4 × 107, about 4 × 107to about 5 × 107, about 5 × 107to about 6 × 107, about 6 × 107to about 7 × 107, about 7 × 107to about 8 × 107, about 8 × 107to about 9 × 107, about 9 × 107to about 1 × 108, about 1 × 108to about 2 × 108, about 2 × 108to about 3 × 108, about 3 × 108to about 4 × 108, about 4 × 108to about 5 × 108, about 5 × 108to about 6 × 108, about 6 × 108to about 7 × 108, about 7 × 108to about 8 × 108, about 8 × 108to about 9 × 108, about 9 × 108to about 1 × 109Tregs are administered to a patient.

[0172] In some embodiments, about 1 × 106to about 2 × 106, about 2 × 106to about 3 × 106, about 3 × 106to about 4 × 106, about 4 × 106to about 5 × 106, about 5 × 106to about 6 × 106, about 6 × 106to about 7 × 106, about 7 × 106to about 8 × 106, about 8 × 106to about 9 × 106, about 9 × 106to about 1 × 107, about 1 × 107to about 2 × 107, about 2 × 107to about 3 × 107, about 3 × 107to about 4 × 107, about 4 × 107to about 5 × 107, about 5 × 107to about 6 × 107, about 6 × 107to about 7 × 107, about 7 × 107to about 8 × 107, about 8 × 107to about 9 × 107, about 9 × 107to about 1 × 108, about 1 × 108to about 2 × 108, about 2 × 108to about 3 × 108, about 3 × 108to about 4 × 108, about 4 × 108to about 5 × 108, about 5 × 108to about 6 × 108, about 6 × 108to about 7 × 108, about 7 × 108to about 8 × 108, about 8 × 108to about 9 × 108, about 9 × 108to about 1 × 109Tregs are administered to a patient in one infusion.

[0173] In some embodiments, a cryopreserved composition comprising a population of Tregs is administered within about 30 minutes, about 1 hour, about 2 to about 3 hours, about 3 to about 4 hours, about 4 to about 5 hours, about 5 to about 6 hours, about 6 to about 7 hours, about 7 to about 8 hours, about 8 to about 9 hours, or about 9 to about 10 hours of thawing the cryopreserved composition comprising a population of Tregs. The cryopreserved composition NAI-1540949621v1 51Atty. Docket No.14678-018-228 comprising a population of Tregs may be stored at about 2 °C to about 8 °C (e.g., at about 4°) between thawing and administration.

[0174] In some embodiments, one dose of a population of Tregs or a composition comprising a population of Tregs is administered to a subject. In some embodiments, a population of Tregs or a composition comprising a population of Tregs is administered more than once. In some embodiments, a population of Tregs or a composition comprising a population of Tregs is administered two or more times. In some embodiments, a population of Tregs or a composition comprising a population of Tregs is administered every 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, 6-7 weeks, 7-8 weeks, 8-9 weeks, 9-10 weeks, 10-11 weeks, 11-12 weeks, every 1-2 months, 2-3 months, 3-4 months, 4-5 months, 5-6 months, 6-7 months, 7-8 months, 8-9 months, 9-10 months, 10-11 months, 11-12 months, 13-14 months, 14-15 months, 15-16 months, 16-17 months, 17-18 months, 18-19 months, 19-20 months, 20-21 months, 21-22 months, 22-23 months, 23-24 months, every 1-2 years, 2-3 years, 3-4 years or 4-5 years.

[0175] In some embodiments, about 1 × 106Tregs per kg of body weight of the subject are administered in the first administration and the number of Tregs administered is increased in the second third and subsequent administration. In some embodiments, about 1 × 106Tregs per kg of body weight of the subject are administered in the first two administrations, and the number of Tregs administered is increased in every other administration thereafter (e.g., the 4th, 6th, 8thand 10thadministration). Thus, for example, about 1 × 106Tregs per kg of body weight of the subject may be administered per month for the first and second month, and about 2 × 106Tregs per kg of body weight of the subject may be administered per month for the third and fourth month, and / or about 3 × 106cells per kg of body weight of the subject are administered per month for the fifth and sixth month.

[0176] In some embodiments, a method of ALS treatment provided herein comprises administering a population of autologous Tregs or a composition comprising a population of autologous Tregs to the subject. In other embodiments, a method of treating ALS in a subject in need thereof comprises administering a population of allogeneic Tregs or a composition comprising a population of allogeneic Tregs to the subject.

[0177] The IL-2 may be administered one, two or more times a month. In some embodiments, the IL-2 is administered three times a month. In some embodiments, the IL-2 is administered subcutaneously. The IL-2 may be administered at least 2 weeks, at least 3 weeks, NAI-1540949621v1 52Atty. Docket No.14678-018-228 or at least 4 weeks prior to the population of anti-inflammatory EVs.

[0178] In certain embodiments of the methods provided herein, a single Treg infusion is administered to the ALS patient. In some embodiments of the methods provided herein, a plurality of Treg infusions is administered to the ALS patient.

[0179] In particular embodiments, the Tregs may be administered with IL-2. In certain embodiments, IL-2 is administered then followed by Treg administration. In some embodiments, Treg administration (e.g., infusion) can be administered concomitantly with IL-2, e.g., with subcutaneous IL-2 injection(s). In some embodiments, the subject is additionally administered IL-2. The dose of IL-2 may be about 0.5-1 × 105IU / m2, about 1-1.5 × 105IU / m2, about 1.5-2 × 105IU / m2, about 2-2.5 × 105IU / m2, about 2.5-3 × 105IU / m2, about 3-3.5 × 105IU / m2, about 3.5-4 × 105IU / m2, about 4-4.5 × 105IU / m2, about 4.5-5 × 105IU / m2, about 5-6 × 105IU / m2, about 6-7 × 105IU / m2, about 7-8 × 105IU / m2, about 8-9 × 105IU / m2, about 9-10 × 105IU / m2, about 10-15 × 105IU / m2, about 15-20 × 105IU / m2, about 20-25 × 105IU / m2, about 25-30 × 105IU / m2, about 30-35 × 105IU / m2, about 35-40 × 105IU / m2, about 40-45 × 105IU / m2, about 45-50 × 105IU / m2, about 50-60 × 105IU / m2, about 60-70 × 105IU / m2, about 70-80 × 105IU / m2, about 80-90 × 105IU / m2, or about 90-100 × 105IU / m2. In specific embodiments, the subject is administered 2 × 105IU / m2of IL-2.

[0180] In some embodiments the ALS therapy comprises anti-inflammatory extracellular vesicles (EVs) derived from ex vivo-expanded Tregs. Exemplary methods for preparing Treg EVs, and for administering Treg EV therapy to patients, are described, for example, in International Application No. PCT / US2022 / 017990, which is incorporated herein in its entirety, in particular for its teaching of such compositions and methods. 7.4.2 IL-2 Therapy

[0181] In certain embodiments of the methods provided herein, the ALS therapy comprises IL-2. In certain embodiments, IL-2 for use in the ALS therapies disclosed herein can be an IL-2 protein (Section 7.4.2(a)). In certain embodiments, methods described herein involve administering to a subject a regimen of low dose IL-2 protein (Section 7.4.2(b)). (a) IL-2 protein

[0182] In certain embodiments, an IL-2 protein for use in the methods described herein can be native IL-2, recombinant IL-2, or synthetic IL-2. Native IL-2 is an antigen-nonspecific, genetically unrestricted soluble factor produced by erythrocyte rosette positive T cells stimulated NAI-1540949621v1 53Atty. Docket No.14678-018-228 with antigens, mitogens, and alloantigens. In some embodiments, IL-2 for use in the methods disclosed herein can comprise a native polypeptide sequence or a variant thereof. A variant of the IL-2 native polypeptide can include a fragment of the IL-2 native polypeptide sequence and / or an analog of the IL-2 native polypeptide sequence, wherein an “analog” of IL-2 refers to a polypeptide comprising the IL-2 native polypeptide sequence comprising one or more amino acid substitutions, insertions, and / or deletions. A variant of the IL-2 native polypeptide can also include any modified IL-2 native polypeptide or fragment or analog thereof, such as, but not limited to, a glycosylated IL-2 native polypeptide or fragment or analog thereof, a phosphorylated IL-2 native polypeptide or fragment or analog thereof, an IL-2 native polypeptide or fragment or analog thereof fused to another polypeptide or molecule, a polymerized IL-2 native polypeptide or fragment or analog thereof, or an IL-2 native polypeptide or fragment or analog thereof subjected to chemical and / or enzymatic modification to improve properties of the IL-2 protein (e.g., stability, specificity, activity, permutation across the blood brain barrier).

[0183] In certain embodiments, the native polypeptide sequence of IL-2 for use in the methods disclosed herein can be obtained from different sources including mammalian sources such as e.g., human, primate, pig, horse, cow, rat, mouse, and the like. In some embodiments, a native polypeptide sequence of IL-2 for use in the methods disclosed herein is a native polypeptide sequence of human IL-2.

[0184] IL-2 proteins are well known. See, e.g., UniProtKB identifier Q0GK43. In another non-limiting example, a nucleotide and an amino acid sequence of human IL-2 are disclosed, for instance, as Gene ID: 3558 in Genbank and UniProt ID: P60568, respectively.

[0185] In certain embodiments, the IL-2 protein is a human IL-2 protein. For example, in certain embodiments, the IL-2 protein is or is derived from the following native mature human IL-2 amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT (SEQ ID NO:1).

[0186] In certain embodiments, the IL-2 protein contains one or more mutations relative to the native mature human IL-2 polypeptide. For example, in certain embodiments, the human IL- 2 protein lacks an N-terminal alanine amino acid. In particular embodiments, the human IL-2 NAI-1540949621v1 54Atty. Docket No.14678-018-228 protein comprises a mutation at the amino acid position corresponding to native mature human IL-2 amino acid residue 125 (underlined in the above sequence). For example, in particular embodiments, the IL-2 protein contains a serine at the amino acid position corresponding to native mature human IL-2 amino acid residue 125. In particular embodiments, the human IL-2 protein lacks an N-terminal alanine amino acid and comprises a serine at the amino acid position corresponding to native mature human IL-2 amino acid residue 125.

[0187] In specific embodiments, the IL-2 protein is not glycosylated.

[0188] In particular embodiments, the human IL-2 protein lacks an N-terminal alanine amino acid, comprises a serine at the amino acid position corresponding to native mature human IL-2 amino acid residue 125, and is not glycosylated.

[0189] In certain embodiments, the IL-2 protein is aldesleukin (des-alanyl-1, serine-125 human interleukin-2; trade name PROLEUKIN), which is well-known. In some embodiments, methods of administering a low-dose IL-2 protein regimen as disclosed herein comprises administration of PROLEUKIN at a dose that is lower than that provided on the PROLEUKIN label.

[0190] In certain embodiments, the human IL-2 protein comprises the following amino acid sequence: PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW ITFSQSIISTLT (SEQ ID NO:2).

[0191] In certain embodiments, the IL-2 protein comprises one or more modifications, e.g., mutations, compared to that of mature human IL-2 and exhibits reduced binding to IL-2 receptor (IL-2R) alpha subunit (CD25) relative to that of native mature IL-2 protein. In certain embodiments, the IL-2 protein comprises one or more modifications, e.g., mutations, compared to that of mature human IL-2 and exhibits reduced binding to Il-2 receptor beta subunit (CD122) relative to that of native IL-2 protein. In certain embodiments, the IL-2 protein comprises one or more modifications, e.g., mutations, compared to that of mature human IL-2 and exhibits reduced binding to IL-2 receptor gamma subunit (CD132) relative to that of wild-type IL-2 protein. In particular embodiments, the IL-2 protein comprises one or more modifications, e.g., mutations, compared to that of mature human IL-2 and exhibits reduced binding to IL-2 receptor beta and gamma subunits relative to that of native mature IL-2 protein. In particular NAI-1540949621v1 55Atty. Docket No.14678-018-228 embodiments, the IL-2 protein comprises one or more modifications, e.g., mutations, compared to that of mature human IL-2 and exhibits reduced binding to IL-2 receptor alpha and beta subunits relative to that of native mature IL-2 protein. In particular embodiments, the IL-2 protein comprises one or more modifications, e.g., mutations, compared to that of mature human IL-2 and exhibits reduced binding to IL-2 receptor beta subunit but not reduced binding to IL-2 receptor subunit alpha relative to that of native mature IL-2 protein. In particular embodiments, the IL-2 protein comprises one or more modifications, e.g., mutations, compared to that of mature human IL-2 and exhibits reduced binding to IL-2 receptor alpha, beta, and gamma subunits relative to that of native mature IL-2 protein.

[0192] In certain embodiments, the IL-2 protein comprises one or more modifications, e.g., mutations, compared to that of mature human IL-2 and exhibits reduced IL-2 receptor-mediated signaling activity relative to that of native mature IL-2 protein. IL-2 receptor-mediated signaling activity may be assayed using routine, well known techniques, for example may be assayed via an assessment of STAT5 phosphorylation. See, e.g., Ghelani et al. (2020) Front. Immunol. 11:1106, which is incorporated herein in its entirety.

[0193] In certain embodiments, the IL-2 protein comprises one or more modifications, e.g., mutations, compared to that of mature human IL-2 and exhibits selectivity for (e.g., preferential activation of) T regulatory (Treg) cells, for example selectivity over natural killer cells and / or T effector cells, as assessed, e.g., by Treg cell proliferation assays, Treg-mediated suppressor function, and / or lineage and / or phenotypic marker expression. Such assays are well-known. See, e.g., Ghelani (2020) Id.

[0194] In some embodiments, the IL-protein is an IL-2 mutein (that is, an IL-2 protein comprising one or more mutations relative to native mature IL-2 protein) comprising an amino acid sequence corresponding to native mature human IL-2, which further comprises a replacement substitution or deletion at one or more amino acid positions, e.g., A1 (deletion); P2 (e.g., deletion); T3 (e.g., T3C, T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P, or deletion); S4 (e.g., deletion); S5 (e.g., deletion); S6 (e.g., deletion); H16 (e.g., H16E, H16R), L18 (e.g., L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18T); D20 (e.g., D20A, D20G, D20H, D20W); Q22 (e.g., Q22F, Q22E, Q22G, Q22A, Q22L, Q22M, Q22F, Q22W, Q22K, Q22S, Q22V, Q22I, Q22Y, Q22H, Q22R, Q22N, Q22D, Q22T, Q22F); K35 (e.g., K35E); R38 (e.g., R38A, R38G); M39 (e.g., M39L, M39V); F42 (e.g., NAI-1540949621v1 56Atty. Docket No.14678-018-228 F42A, F42L, F42Y); Y45 (e.g., Y45A); H55 (e.g., H55Y); C58 (e.g., deletion); E61 (e.g., E61Q); E62 (e.g., E62A); I86 (e.g., I86V); N88 (e.g., N88I, N88G, N88D, N88K, N88R); I89 (e.g., I89V); V91 (e.g., V91D, V91K); I92 (e.g., I92F); K97 (e.g., K97Q); M104 (e.g., M104A, M104T, M104V); D109 (e.g., D109C or substituted with a non-natural amino acid with an activated side chain); T113 (e.g., T113N); C125 (e.g., C125A, C125S); Q126 (e.g., Q126H, Q126M, Q126K, Q126C, Q126D, Q126E, Q126G, Q126I, Q126R, Q126S, Q126T); or S130 (e.g., S130T, S130G, S130R).

[0195] In certain embodiments, the human IL-2 protein comprises the following amino acid sequence: PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQLEE ELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWI TFSQSIISTLT (SEQ ID NO:3).

[0196] In some embodiments, the IL-protein is an IL-2 mutein that comprises an amino acid sequence corresponding to native mature human IL-2 and which further comprises D20A and H16E mutations; D20A and M104T mutations; H16E and E61Q mutations; V91K, D20A and M104V mutations; a D20G mutation; a D20W mutation; a F42Y mutation; a N88K mutation; or D20A, H16R and E61Q mutations.

[0197] In some embodiments, the IL-protein is an IL-2 mutein that comprises an amino acid sequence corresponding to native mature human IL-2 and which further comprises a D20, N88 and / or Q126 mutation. For example, in certain embodiments, an IL-2 protein may comprise a D20H mutation, a N88R, N88I or N88G mutation, and / or a Q126D mutation. See, e.g., U.S. Patent No.6,955,807.

[0198] In some embodiments, the IL-2 protein is an IL-2Ra / IL-2Rb biased IL-2 protein. For example, in certain embodiments, the IL-12 is STK-012 (Emmerich, J. et al. Cancer Res 2021;81(13_Suppl):Abstract nr 1744).

[0199] In particular embodiments, the IL-protein is an IL-2 mutein that comprises an amino acid sequence corresponding to native mature human IL-2 and which further comprises mutations at one or more of L53 (e.g., L53I), L56 (e.g., L56I), L80 (e.g., L80I), and L118 (e.g., L118I); and, optionally, mutations at one or more of V69 (e.g., V69A), Q74 (e.g., Q74P), N88 (e.g., N88D), and C125 (e.g., C125S), for example, L53I, N88D, V69A, Q74P, and C125S mutations; L56I, N88D, V69A, Q74P, and C125S mutations; L80I, N88D, V69A, Q74P, and NAI-1540949621v1 57Atty. Docket No.14678-018-228 C125S mutations; or L118I, N88D, V69A, Q74P, and C125S mutations.

[0200] In some embodiments, the IL-2 protein is an IL-2 mutein that comprises an amino acid sequence corresponding to native mature human IL-2, which further comprises a replacement substitution at one or more positions: E15 (e.g., E15Q); H16 (e.g., H16N); Q22 (e.g., Q22E); N29 (e.g., N29S); Y31 (e.g., Y31S, Y31H); K35 (e.g., K35R); T37 (e.g., T37A); K48 (e.g., K48E); V69 (e.g., V69A); N71 (e.g., N71R); Q74 (e.g., Q74P); D84 (e.g., D84N); N88 (e.g., N88D, N88R); E95 (e.g., E95Q); C125 (e.g., C125A, C125S); or Q126 (e.g., Q126E).

[0201] In certain embodiments, an IL-2 protein for use in the methods described herein can be a recombinant IL-2 protein. Any suitable method of generating recombinant proteins known in the art can be used to generate the recombinant IL-2 protein for use in any of the methods disclosed herein. Such methods can include those described in, for example, Lundblad and Macdonald (2018) HANDBOOK OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (5th ed), CRC Press, and Green and Sambrook (2012) MOLECULAR CLONING: A LABORATORY MANUAL (4thed). Cold Spring Harbor Laboratory Press. In certain embodiments, a host organism can be used to generate a recombinant IL-2 protein for use in any of the methods disclosed herein. In some embodiments, a host organism can be a prokaryotic host (e.g., a bacterium such as E. coli) and / or an eukaryotic host (e.g., a yeast, fungus, plant, or mammalian cell). In certain embodiments, an IL-2 protein for use in the methods described herein can be a recombinant human IL-2 protein.

[0202] In certain embodiments, an IL-2 protein for use in the methods described herein can be administered as part of a formulation. In some embodiments, a formulation comprising an IL- 2 protein for use in the methods disclosed herein can include at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients as described herein can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Non-limiting examples of IL-2 formulations are described in U.S. Patent No.4,604,377, the disclosure of which is incorporated herein in its entirety, in particular for its teaching of such IL-2 formulations.

[0203] In certain embodiments, an IL-2 protein for use in the methods described herein can be aldesleukin. Aldesleukin is a recombinant form of human IL-2 and is the active ingredient in formulations commercially known as Proleukin®or Roncoleukin®. In some embodiments, methods of administering a low-dose IL-2 protein regimen as disclosed herein comprise NAI-1540949621v1 58Atty. Docket No.14678-018-228 administration of Proleukin®IL-2. In some embodiments, methods of administering a low-dose IL-2 protein regimen as disclosed herein comprise administration of Proleukin®IL-2 at a dose that is lower than that provided on the Proleukin®label. (b) Regimen of Low Dose IL-2 Protein Administration

[0204] In certain embodiments, methods described herein involve administering to a subject a regimen of low dose IL-2 protein. In certain embodiments a subject is a human subject. In certain embodiments, a subject for administration of a regimen of low dose IL-2 may be diagnosed with or suspected of having ALS. In any of the embodiments presented herein, the IL-2 protein may be aldesleukin.

[0205] It will be understood that the standard quantitative measure for IL-2 is the International Unit (IU), which is based not on mass of protein but on activity in a biological assay such as that as established by the World Health Organization 1st International Standard for Interleukin-2 (human). However, in practice, when manufacture of an IL-2 product is standardized, a conversion between drug mass and units is routinely possible. For the PROLEUKIN (aldesleukin) product, for example, the conversion is 18 million international units (18×106IU) equal to 1.1 mg IL-2 protein.

[0206] In certain embodiments, a patient disclosed herein can be administered less than 0.30 mg IL-2 protein. In certain embodiments, a patient disclosed herein can be administered an amount of IL-2 protein ranging from about 0.0005 mg to about 0.30 mg, from about 0.0006 mg to about 0.30 mg, from about 0.0007 mg to about 0.30 mg, from about 0.0008 mg to about 0.30 mg, from about 0.0009 mg to about 0.30 mg, from about 0.001 mg to about 0.30 mg, from about 0.005 mg to about 0.30 mg, from about 0.01 mg to about 0.30 mg, from about 0.05 mg to about 0.30 mg, or from about 0.1 mg to about 0.30 mg. In certain embodiments, a patient disclosed herein can be administered about 0.0005 mg IL-2 protein, about 0.0006 mg IL-2 protein, about 0.0007 mg IL-2 protein, about 0.0008 mg IL-2 protein, about 0.0009 mg IL-2 protein, about 0.001 mg IL-2 protein, about 0.0025 mg IL-2 protein, about 0.005 mg IL-2 protein, about 0.0075 mg IL-2 protein, about 0.01 mg IL-2 protein, about 0.02 mg IL-2 protein, about 0.025 mg IL-2 protein, about 0.03 mg IL-2 protein, about 0.04 mg IL-2 protein, about 0.05 mg IL-2 protein, about 0.075 mg IL-2 protein, about 0.1 mg IL-2 protein, about 0.15 mg IL-2 protein, about 0.175 mg IL-2 protein, about 0.2 mg IL-2 protein, about 0.225 mg IL-2 protein, about 0.25 mg IL-2 protein, about 0.275 mg IL-2 protein, about 0.28 mg IL-2 protein, about 0.29 mg IL-2 protein, NAI-1540949621v1 59Atty. Docket No.14678-018-228 about 0.3 mg IL-2 protein, or about 0.35 mg IL-2 protein.

[0207] In certain embodiments, a patient can be administered a dosage of IL-2 below about 9,000,000 (9 ×106) IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 8.7 ×106IU. In certain embodiments, a patient can be administered a dosage of IL- 2 below about 8.3 ×106IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 8.0 ×106IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 7.8 ×106IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 7.5 ×106IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 7.0 ×106IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 6.5 ×106IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 6.0 ×106IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 5.7 ×106IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 5.5 ×106IU. In certain embodiments, a patient can be administered a dosage of IL-2 below about 5.2 ×106IU. In certain embodiments, a patient disclosed herein can be administered less than about 5×106IU IL-2 protein. In certain embodiments, a patient disclosed herein can be administered an amount of IL- 2 protein ranging from about 0.1 ×106IU to about 5 ×106IU, from about 0.2 ×106IU to about 5 ×106IU, from about 0.3 ×106IU to about 5 ×106IU, from about 0.4 ×106IU to about 5 ×106IU, from about 0.5 ×106IU to about 5 ×106IU, from about 1 ×106IU to about 5 ×106IU, from about 1.5 ×106IU to about 5 ×106IU, from about 2 ×106IU to about 5 ×106IU, from about 2.5 ×106IU to about 5 ×106IU, from about 3 ×106IU to about 5 ×106IU, from about 3.5 ×106IU to about 5 ×106IU, from about 4 ×106IU to about 5 ×106IU, from about 4.5 ×106IU to about 5 ×106IU, from about 0.1 ×106IU to about 4 ×106IU, from about 0.2 ×106IU to about 4 ×106IU, from about 0.3 ×106IU to about 4 ×106IU, from about 0.4 ×106IU to about 4 ×106IU, from about 0.5 ×106IU to about 4 ×106IU, from about 1 ×106IU to about 4 ×106IU, from about 1.5 ×106IU to about 4 ×106IU, from about 2 ×106IU to about 4 ×106IU, from about 2.5 ×106IU to about 4 ×106IU, from about 3 ×106IU to about 4 ×106IU, from about 3.5 ×106IU to about 4 ×106IU, from about 0.1 ×106IU to about 3 ×106IU, from about 0.2 ×106IU to about 3 ×106IU, from about 0.3 ×106IU to about 3 ×106IU, from about 0.4 ×106IU to about 3 ×106IU, from about 0.5 ×106IU to about 3 ×106IU, from about 1 ×106IU to about 3 ×106IU, from about 1.5 ×106IU to about 3 ×106IU, from about 2 ×106IU to about 3 ×106IU, from about 2.5 ×106IU to about 3 NAI-1540949621v1 60Atty. Docket No.14678-018-228 ×106IU, from about 0.1 ×106IU to about 2 ×106IU, from about 0.2 ×106IU to about 2 ×106IU, from about 0.3 ×106IU to about 2 ×106IU, from about 0.4 ×106IU to about 2 ×106IU, from about 0.5 ×106IU to about 2 ×106IU, from about 1 ×106IU to about 2 ×106IU, from about 1.5 ×106IU to about 2 ×106IU, from about 0.1 ×106IU to about 1 ×106IU, from about 0.2 ×106IU to about 1 ×106IU, from about 0.3 ×106IU to about 1 ×106IU, from about 0.4 ×106IU to about 1 ×106IU, or from about 0.5 ×106IU to about 1 ×106IU IL-2 protein.

[0208] In certain embodiments, a patient disclosed herein can be administered about 0.1×106IU, about 0.2×106IU, about 0.25×106IU, about 0.3×106IU, about 0.4×106IU, about 0.5×106IU, about 0.6×106IU, about 0.7×106IU, about 0.75×106IU, about 1×106IU, about 0.8×106IU, about 0.9×106IU, about 1.0×106IU, about 1.25×106IU, about 1.5×106IU, about 1.75×106IU, about 2×106IU, about 2.25×106IU, about 2.5×106IU, about 2.75×106IU, about 3×106IU, about 3.25×106IU, about 3.5×106IU, about 3.75×106IU, about 4×106IU, about 4.25×106IU, about 4.5×106IU, about 4.75×106IU, or about 5×106IU, about 5.2×106IU, about 5.6×106IU, about 6.0×106IU, about 6.5×106IU, about 7.2×106IU, about 7.4×106IU, about 7.8×106IU, about 8.0×106IU, about 8.2×106IU, about 8.5×106IU, about 8.6×106IU, about 8.7×106IU, about 8.8×106IU, about 8.9×106IU, or about 9.0×106IU IL-2 protein.

[0209] In certain embodiments, a patient can be administered a dosage of IL-2 below about 9 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 8.7 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 8.3 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 8.0 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 7.8 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 7.5 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 7.0 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 6.5 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 6.0 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 5.7 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 5.5 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 5.2 ×106IU / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 5 ×106IU / day. In certain embodiments, a patient can be administered a dosage of NAI-1540949621v1 61Atty. Docket No.14678-018-228 IL-2 below at least about 5 ×106IU / day. In some embodiments, a patient can be administered a dosage of IL-2 ranging from about 0.1 ×106IU / day to about 5 ×106IU / day, from about 0.2 ×106IU / day to about 5 ×106IU / day, from about 0.3 ×106IU / day to about 5 ×106IU / day, from about 0.4 ×106IU / day to about 5 ×106IU / day, from about 0.5 ×106IU / day to about 5 ×106IU / day, from about 1 ×106IU / day to about 5 ×106IU / day, from about 1.5 ×106IU / day to about 5 ×106IU / day, from about 2 ×106IU / day to about 5 ×106IU / day, from about 2.5 ×106IU / day to about 5 ×106IU / day, from about 3 ×106IU / day to about 5 ×106IU / day, from about 3.5 ×106IU / day to about 5 ×106IU / day, from about 4 ×106IU / day to about 5 ×106IU / day, from about 4.5 ×106IU / day to about 5 ×106IU / day, from about 0.1 ×106IU / day to about 4 ×106IU / day, from about 0.2 ×106IU / day to about 4 ×106IU / day, from about 0.3 ×106IU / day to about 4 ×106IU / day, from about 0.4 ×106IU / day to about 4 ×106IU / day, from about 0.5 ×106IU / day to about 4 ×106IU / day, from about 1 ×106IU / day to about 4 ×106IU / day, from about 1.5 ×106IU / day to about 4 ×106IU / day, from about 2 ×106IU / day to about 4 ×106IU / day, from about 2.5 ×106IU / day to about 4 ×106IU / day, from about 3 ×106IU / day to about 4 ×106IU / day, from about 3.5 ×106IU / day to about 4 ×106IU / day, from about 0.1 ×106IU / day to about 3 ×106IU / day, from about 0.2 ×106IU / day to about 3 ×106IU / day, from about 0.3 ×106IU / day to about 3 ×106IU / day, from about 0.4 ×106IU / day to about 3 ×106IU / day, from about 0.5 ×106IU to about 3 ×106IU / day, from about 1 ×106IU / day to about 3 ×106IU / day, from about 1.5 ×106IU / day to about 3 ×106IU / day, from about 2 ×106IU / day to about 3 ×106IU / day, from about 2.5 ×106IU / day to about 3 ×106IU / day, from about 0.1 ×106IU / day to about 2 ×106IU / day, from about 0.2 ×106IU / day to about 2 ×106IU / day, from about 0.3 ×106IU / day to about 2 ×106IU / day, from about 0.4 ×106IU / day to about 2 ×106IU / day, from about 0.5 ×106IU / day to about 2 ×106IU / day, from about 1 ×106IU / day to about 2 ×106IU / day, from about 1.5 ×106IU / day to about 2 ×106IU / day, from about 0.1 ×106IU / day to about 1 ×106IU / day, from about 0.2 ×106IU / day to about 1 ×106IU / day, from about 0.3 ×106IU / day to about 1 ×106IU / day, from about 0.4 ×106IU / day to about 1 ×106IU / day, or from about 0.5 ×106IU / day to about 1 ×106IU / day of IL-2 protein.

[0210] In certain embodiments, a patient can be administered a dosage of IL-2 less than about 0.1 ×106IU / day to about 5 ×106IU / day, about 0.25 ×106IU / day to about 4.75 ×106IU / day, about 0.5 ×106IU / day to about 4.5 ×106IU / day, or about 0.75 ×106IU / day to about 4.0 ×106IU / day. In some embodiments, a patient can be administered a dosage of IL-2 less than about 0.1 ×106IU / day, less than about 0.25 ×106IU / day, less than about 0.5 ×106IU / day, less NAI-1540949621v1 62Atty. Docket No.14678-018-228 than about 0.75 ×106IU / day, less than about 1.0 ×106IU / day, less than about 1.25 ×106IU / day, less than about 1.5 ×106IU / day, less than about 1.75 ×106IU / day, less than about 2.0 ×106IU / day, less than about 2.25 ×106IU / day, less than about 2.5 ×106IU / day, less than about 2.75 ×106IU / day, less than about 3.0 ×106IU / day, less than about 3.25 ×106IU / day, less than about 3.5 ×106IU / day, less than about 3.75 ×106IU / day, less than about 4.0 ×106IU / day, less than about 4.25 ×106IU / day, less than about 4.5 ×106IU / day, less than about 4.75 ×106IU / day, or less than about 5.0 ×106IU / day. In some embodiments, a patient can be administered a dosage of IL-2 at about 0.1 ×106IU / day, at about 0.25 ×106IU / day, at about 0.5 ×106IU / day, at about 0.75 ×106IU / day, at about 1.0 ×106IU / day, at about 1.25 ×106IU / day, at about 1.5 ×106IU / day, at about 1.75 ×106IU / day, at about 2.0 ×106IU / day, at about 2.25 ×106IU / day, at about 2.5 ×106IU / day, at about 2.75 ×106IU / day, at about 3.0 ×106IU / day, at about 3.25 ×106IU / day, at about 3.5 ×106IU / day, at about 3.75 ×106IU / day, at about 4.0 ×106IU / day, at about 4.25 ×106IU / day, at about 4.5 ×106IU / day, at about 4.75 ×106IU / day, or at about 5.0 ×106IU / day.

[0211] In certain embodiments, a patient disclosed herein can be administered less than about 0.1×106IU / day, less than about 0.2×106IU / day, less than about 0.25×106IU / day, less than about 0.3×106IU / day, less than about 0.4×106IU / day, less than about 0.5×106IU / day, less than about 0.6×106IU / day, less than about 0.7×106IU / day, less than about 0.75×106IU / day, less than about 0.8×106IU / day, less than about 0.9×106IU / day, less than about 1.0×106IU / day, less than about 1.25×106IU / day, less than about 1.5×106IU / day, less than about 1.75×106IU / day, less than about 2×106IU / day, less than about 2.25×106IU / day, less than about 2.5×106IU / day, less than about 2.75×106IU / day, less than about 3×106IU / day, less than about 3.25×106IU / day, less than about 3.5×106IU / day, less than about 3.75×106IU / day, less than about 4×106IU / day, less than about 4.25×106IU / day, less than about 4.5×106IU / day, less than about 4.75×106IU / day, or less than about 5×106IU / day of IL-2 protein.

[0212] In certain embodiments, a patient disclosed herein can be administered about 0.1×106IU / day, about 0.2×106IU / day, about 0.25×106IU / day, about 0.3×106IU / day, about 0.4×106IU / day, about 0.5×106IU / day, about 0.6×106IU / day, about 0.7×106IU / day, about 0.75×106IU / day, about 1×106IU / day, about 0.8×106IU / day, about 0.9×106IU / day, about 1.0×106IU / day, about 1.25×106IU / day, about 1.5×106IU / day, about 1.75×106IU / day, about 2×106IU / day, about 2.25×106IU / day, about 2.5×106IU / day, about 2.75×106IU / day, about 3×106IU / day, about 3.25×106IU / day, about 3.5×106IU / day, about 3.75×106IU / day, about 4×106IU / day, NAI-1540949621v1 63Atty. Docket No.14678-018-228 about 4.25×106IU / day, about 4.5×106IU / day, about 4.75×106IU / day, about 5×106IU / day, about 5.2×106IU / day, about 5.4×106IU / day, about 5.6×106IU / day, about 5.8×106IU / day, about 6.0×106IU / day, about 6.5×106IU / day, about 7.2×106IU / day, about 7.4×106IU / day, about 7.8×106IU / day, about 8.0×106IU / day, about 8.2×106IU / day, about 8.5×106IU / day, about 8.6×106IU / day, about 8.7×106IU / day, about 8.8×106IU / day, about 8.9×106IU / day, or about 9.0×106IU / day IL-2 protein.

[0213] In certain embodiments, the amount of IL-2 administered to a patient can be determined from the weight of the patient. One of skill in the art can appreciate that body mass varies from subject to subject based on age, gender, and / or geographic location. In some embodiments, the amount of IL-2 administered to a human patient can be based on the patient’s body mass (kg), wherein the average body mass of a human ranges from about 60 kg to about 80 kg (see, e.g., Walpole et al., BMC Public Health.2012 Jun 18;12:439). In certain embodiments, a patient can be administered a dosage of IL-2 below about 0.3 ×106IU / kg / day. In certain embodiments, a patient can be administered a dosage of IL-2 below at least about 0.2 ×106IU / kg / day. In certain embodiments, a patient can be administered a dosage of IL-2 below about 0.15 ×106IU / kg / day. In certain embodiments, a patient can be administered a dosage of IL-2 below at least about 0.13 ×106IU / kg / day. In certain embodiments, a patient can be administered a dosage of IL-2 below at least about 0.1 ×106IU / kg / day. In some embodiments, a patient can be administered a dosage of IL-2 ranging from about 0.001 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.002 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.0025 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.003 ×106IU / kg / day to about 0.13 ×106IU / kg / day, 0.004 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.005 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.006 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.008 ×106IU / kg / day to about 0.13 ×106IU / kg / day, 0.009 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.010 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.012 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.014 ×106IU / kg / day to about 0.13 ×106IU / kg / day, 0.0181 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.02 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.025 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.03 ×106IU / kg / day to about 0.13 ×106IU / kg / day, 0.035 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.0375 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.04 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.05 ×106IU / kg / day to about 0.13 ×106NAI-1540949621v1 64Atty. Docket No.14678-018-228 IU / kg / day, 0.055 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.060 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.065 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.070 ×106IU / kg / day to about 0.13 ×106IU / kg / day, 0.075 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.080 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.0855 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.090 ×106IU / kg / day to about 0.13 ×106IU / kg / day, 0.0925 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.095 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.1 ×106IU / kg / day to about 0.105 ×106IU / kg / day, about 0.11 ×106IU / kg / day to about 0.115 ×106IU / kg / day, 0.175 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.12 ×106IU / kg / day to about 0.13 ×106IU / kg / day, about 0.125 ×106IU / kg / day to about 0.13 ×106IU / kg / day, or about 0.013 ×106IU / kg / day to about 0.13 ×106IU / kg / day IL-2 protein.

[0214] In some embodiments, a patient can be administered a dosage of IL-2 less than about 0.001 ×106IU / kg / day, less than about 0.002 ×106IU / kg / day, less than about 0.0025 ×106IU / kg / day, less than about 0.003 ×106IU / kg / day, less than about 0.004 ×106IU / kg / day, less than about 0.005 ×106IU / kg / day, less than about 0.006 ×106IU / kg / day, less than about 0.008 ×106IU / kg / day, less than about 0.009 ×106IU / kg / day, less than about 0.010 ×106IU / kg / day, less than about 0.012 ×106IU / kg / day, less than about 0.014 ×106IU / kg / day, less than about 0.0181 ×106IU / kg / day, less than about 0.02 ×106IU / kg / day, less than about 0.025 ×106IU / kg / day, less than about 0.03 ×106IU / kg / day, less than about 0.035 ×106IU / kg / day, less than about 0.0375 ×106IU / kg / day, less than about 0.04 ×106IU / kg / day, less than about 0.05 ×106IU / kg / day, less than about 0.055 ×106IU / kg / day, less than about 0.060 ×106IU / kg / day, less than about 0.065 ×106IU / kg / day, less than about 0.070 ×106IU / kg / day, less than about 0.075 ×106IU / kg / day, less than about 0.080 ×106IU / kg / day, less than about 0.0855 ×106IU / kg / day, less than about 0.090 ×106IU / kg / day, less than about 0.0925 ×106IU / kg / day, less than about 0.095 ×106IU / kg / day, less than about 0.1 ×106IU / kg / day, less than about 0.11 ×106IU / kg / day, less than about 0.115 ×106IU / kg / day, less than about 0.175 ×106IU / kg / day, less than about 0.12 ×106IU / kg / day, less than about 0.125 ×106IU / kg / day, or less than about 0.013 ×106IU / kg / day IL-2 protein.

[0215] In some embodiments, a patient can be administered a dosage of IL-2 about 0.001 ×106IU / kg / day, about 0.002 ×106IU / kg / day, about 0.0025 ×106IU / kg / day, about 0.003 ×106IU / kg / day, about 0.004 ×106IU / kg / day, about 0.005 ×106IU / kg / day, about 0.006 ×106IU / kg / day, about 0.008 ×106IU / kg / day, about 0.009 ×106IU / kg / day, about 0.010 ×106NAI-1540949621v1 65Atty. Docket No.14678-018-228 IU / kg / day, about 0.012 ×106IU / kg / day, about 0.014 ×106IU / kg / day, about 0.0181 ×106IU / kg / day, about 0.02 ×106IU / kg / day, about 0.025 ×106IU / kg / day, about 0.03 ×106IU / kg / day, about 0.035 ×106IU / kg / day, about 0.0375 ×106IU / kg / day, about 0.04 ×106IU / kg / day, about 0.05 ×106IU / kg / day, about 0.055 ×106IU / kg / day, about 0.060 ×106IU / kg / day, about 0.065 ×106IU / kg / day, about 0.070 ×106IU / kg / day, about 0.075 ×106IU / kg / day, about 0.080 ×106IU / kg / day, about 0.0855 ×106IU / kg / day, about 0.090 ×106IU / kg / day, about 0.0925 ×106IU / kg / day, about 0.095 ×106IU / kg / day, about 0.1 ×106IU / kg / day, about 0.11 ×106IU / kg / day, about 0.115 ×106IU / kg / day, about 0.175 ×106IU / kg / day, about 0.12 ×106IU / kg / day, about 0.125 ×106IU / kg / day, or about 0.013 ×106IU / kg / day IL-2 protein.

[0216] In certain embodiments, the amount of IL-2 administered to a patient can be determined from the body surface area (BSA) of the patient. Any method known in the art for determining the BSA of the human body can be used to determine IL-2 dosing for the methods disclosed herein. In certain embodiments, BSA can be calculated using the DuBois and DuBois formula (see Dubois and Dubois, Arch Intern Med.1916; 17:863-871) according to equation I: BSA (m2) = 0.007184 × height (cm)0.725 × weight (kg)0.425 (I).

[0217] In certain embodiments, BSA can be calculated using the Gehan and George formula (see Gehan and George, Cancer Chemother Rep.1970; 54:225) according to equation II: BSA (m2) = 0.0235 × height (cm)0.42246 × weight (kg)0.51456 (II).

[0218] In certain embodiments, BSA can be calculated using the Haycock formula (see Haycock et al., J Pediatr.1978; 93:62) according to equation III: BSA (m2) = 0.024265 × height (cm)0.3964 × weight (kg)0.5378 (III).

[0219] In certain embodiments, BSA can be calculated using the Mosteller formula (see Mosteller, N Engl J Med.1987; 317:1098) according to equation IV: BSA (m2) = √ ((height (cm) × weight (kg) / 3600) (IV).

[0220] Examples of average BSA values include, but are not limited to: infant, 0.25 m2; child (~10 years), 1.14 m2; adult male, 1.9 m2; and adult female, 1.6 m2. In some embodiments, the average adult is 1.73 m2.

[0221] In certain embodiments, a patient can be administered a regimen of low dose IL-2 wherein the dose of IL-2 is below at least about 5 ×106IU / m2 / day. In some embodiments, a NAI-1540949621v1 66Atty. Docket No.14678-018-228 patient can be administered a regimen of low dose IL-2 wherein the dose of IL-2 ranges from about 0.1 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 0.2 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 0.3 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 0.4 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 0.5 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 0.6 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 0.7 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 0.8 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 0.9 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 01.0 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 1.5 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 2.0 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 2.5 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 3.0 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 3.5 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 4.0 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 4.5 ×106IU / m2 / day to about 5 ×106IU / m2 / day, about 0.1 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 0.2 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 0.3 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 0.4 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 0.5 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 0.6 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 0.7 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 0.8 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 0.9 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 01.0 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 1.5 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 2.0 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 2.5 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 3.0 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 3.5 ×106IU / m2 / day to about 4 ×106IU / m2 / day, about 0.1 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 0.2 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 0.3 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 0.4 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 0.5 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 0.6 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 0.7 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 0.8 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 0.9 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 01.0 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 1.5 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 2.0 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 2.5 ×106IU / m2 / day to about 3 ×106IU / m2 / day, about 0.1 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 0.2 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 0.3 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 0.4 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 0.5 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 0.6 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 0.7 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 0.8 ×106IU / m2 / day to NAI-1540949621v1 67Atty. Docket No.14678-018-228 about 2 ×106IU / m2 / day, about 0.9 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 01.0 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 1.5 ×106IU / m2 / day to about 2 ×106IU / m2 / day, about 0.1 ×106IU / m2 / day to about 1 ×106IU / m2 / day, about 0.2 ×106IU / m2 / day to about 1 ×106IU / m2 / day, about 0.3 ×106IU / m2 / day to about 1 ×106IU / m2 / day, about 0.4 ×106IU / m2 / day to about 1 ×106IU / m2 / day, about 0.5 ×106IU / m2 / day to about 1 ×106IU / m2 / day, about 0.6 ×106IU / m2 / day to about 1 ×106IU / m2 / day, about 0.7 ×106IU / m2 / day to about 1 ×106IU / m2 / day, about 0.8 ×106IU / m2 / day to about 1 ×106IU / m2 / day, about 0.9 ×106IU / m2 / day to about 1 ×106IU / m2 / day, about 0.25 ×106IU / m2 / day to about 4.75 ×106IU / m2 / day, about 0.5 ×106IU / m2 / day to about 4.5 ×106IU / m2 / day, or about 0.75 ×106IU / m2 / day to about 4.0 ×106IU / m2 / day.

[0222] In some embodiments, a patient can be administered a regimen of low dose IL-2 wherein the dose of IL-2 is less than about 0.1 ×106IU / m2 / day, less than about 0.2 ×106IU / m2 / day, less than about 0.25 ×106IU / m2 / day, less than about 0.3 ×106IU / m2 / day, less than about 0.4 ×106IU / m2 / day, less than about 0.5 ×106IU / m2 / day, less than about 0.6 ×106IU / m2 / day, less than about 0.7 ×106IU / m2 / day, less than about 0.75 ×106IU / m2 / day, less than about 0.8 ×106IU / m2 / day, less than about 0.9 ×106IU / m2 / day, less than about 1.0 ×106IU / m2 / day, less than about 1.25 ×106IU / m2 / day, less than about 1.5 ×106IU / m2 / day, less than about 1.75 ×106IU / m2 / day, less than about 2.0 ×106IU / m2 / day, less than about 2.25 ×106IU / m2 / day, less than about 2.5 ×106IU / m2 / day, less than about 2.75 ×106IU / m2 / day, less than about 3.0 ×106IU / m2 / day, less than about 3.25 ×106IU / m2 / day, less than about 3.5 ×106IU / m2 / day, less than about 3.75 ×106IU / m2 / day, less than about 4.0 ×106IU / m2 / day, less than about 4.25 ×106IU / m2 / day, less than about 4.5 ×106IU / m2 / day, less than about 4.75 ×106IU / m2 / day, or less than about 5.0 ×106IU / m2 / day.

[0223] In some embodiments, a patient can be administered a regimen of low dose IL-2 wherein the dose of IL-2 is about 0.1 ×106IU / m2 / day, about 0.2 ×106IU / m2 / day, about 0.25 ×106IU / m2 / day, about 0.3 ×106IU / m2 / day, about 0.4 ×106IU / m2 / day, about 0.5 ×106IU / m2 / day, about 0.6 ×106IU / m2 / day, about 0.7 ×106IU / m2 / day, about 0.75 ×106IU / m2 / day, about 0.8 ×106IU / m2 / day, about 0.9 ×106IU / m2 / day, about 1.0 ×106IU / m2 / day, about 1.1 ×106IU / m2 / day, about 1.2 ×106IU / m2 / day, about 1.25 ×106IU / m2 / day, about 1.3 ×106IU / m2 / day, about 1.4 ×106IU / m2 / day, about 1.5 ×106IU / m2 / day, about 1.6 ×106IU / m2 / day, about 1.7 ×106IU / m2 / day, about 1.75 ×106IU / m2 / day, about 1.8 ×106IU / m2 / day, about 1.9 ×106IU / m2 / day, about 2.0 ×106IU / m2 / day, about 2.0 ×106IU / m2 / day, about 2.1 ×106IU / m2 / day, about 2.2 ×106IU / m2 / day, NAI-1540949621v1 68Atty. Docket No.14678-018-228 about 2.25 ×106IU / m2 / day, about 2.5 ×106IU / m2 / day, about 2.75 ×106IU / m2 / day, about 3.0 ×106IU / m2 / day, about 3.25 ×106IU / m2 / day, about 3.5 ×106IU / m2 / day, about 3.75 ×106IU / m2 / day, about 4.0 ×106IU / m2 / day, at about 4.25 ×106IU / m2 / day, about 4.5 ×106IU / m2 / day, about 4.75 ×106IU / m2 / day, or about 5.0 ×106IU / m2 / day.

[0224] In certain embodiments, a regimen of low dose IL-2 administered to the patient as described herein can be administered to the patient by injection or infusion. In certain embodiments, a regimen of low dose IL-2 administered to the patient as described herein can be administered to the patient intravenously. In certain embodiments, a regimen of low dose IL-2 administered to the patient as described herein can be administered to the patient subcutaneously. 7.4.3 IL-2 and CTLA-4 Fusion Protein Therapy

[0225] In some embodiments, the ALS therapy is IL-2 and CTLA-4 fusion protein, e.g., abatacept. Exemplary methods of administering IL-2 and abatacept combination therapy are described, for example, in International Application No. PCT / US2022 / 019748, which is incorporated herein by reference for its teaching of such methods. In some embodiments, the ALS therapy can comprise a CTLA-4-containing protein and an IL-2 protein. Any of the IL-2 proteins disclosed in Section 7.4.2(a) are suited for use in an ALS therapy comprising IL-2 and a CTLA-4-containing protein. In certain embodiments, the CTLA-4-containing protein is abatacept. In certain embodiments, the IL-2 protein is aldesleukin. In certain embodiments, the CTLA-4-containing protein is abatacept and the IL-2 protein is aldesleukin.

[0226] In certain embodiments, the CTLA-4-containing protein comprises a human CTLA-4 extracellular domain. In particular embodiments, the CTLA-4-containing protein is a fusion protein, for example, a fusion protein that comprises a human CTLA-4 extracellular domain and a human immunoglobulin Fc domain, e.g., a modified Fc domain that comprises an immunoglobulin hinge region, CH2 region and CH3. In particular embodiments, the human immunoglobulin Fc domain is a human IgG1 Fc domain. In certain embodiments, the CTLA-4- containing protein is glycosylated.

[0227] In certain embodiments, the CTLA-4-containing protein comprises the following amino acid sequence monomer: MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNE LTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPE PCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE NAI-1540949621v1 69Atty. Docket No.14678-018-228 DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO:4). In specific embodiments, the CTLA-4-containing protein comprises a homodimer of two monomers, each monomer comprising the amino acid sequence of SEQ ID NO:4. In particular embodiments, the CTLA-4-containing protein is abatacept.

[0228] In some embodiments, according to the methods provided herein, the CTLA-4- containing protein is administered to the subject once every two weeks. In some embodiments, the CTLA-4-containing protein is administered intravenously to the subject once every two weeks. In some embodiments, the CTLA-4-containing protein is administered subcutaneously to the subject once every two weeks. In some embodiments, the CTLA-4-containing protein is administered to the subject once every two weeks for 10-20 weeks. For example, in some embodiments, the CTLA-4-containing protein is administered to the subject once every two weeks for 12 weeks, for 15 weeks, or for 18 weeks. In some embodiments, the CTLA-4- containing protein is abatacept.

[0229] In some embodiments, according to the methods provided herein, the CTLA-4- containing protein is administered to the subject once every week. In some embodiments, the CTLA-4-containing protein is administered intravenously to the subject once every week. In some embodiments, the CTLA-4-containing protein is administered subcutaneously to the subject once every week. In some embodiments, the CTLA-4-containing protein is administered to the subject once every week for 10-20 weeks. For example, in some embodiments, the CTLA-4-containing protein is administered to the subject once week for 12 weeks, for 15 weeks, or for 18 weeks. In some embodiments, the CTLA-4-containing protein is abatacept.

[0230] In some embodiments, according to the methods provided herein, the CTLA-4- containing protein is administered to the subject once daily for 2-5 consecutive days. For example, in some embodiments, the CTLA-4-containing protein is administered to the subject once daily for 2, 3, 4 or 5 consecutive days. In some embodiments, the CTLA-4-containing protein is administered to the subject once daily for three consecutive days. In some embodiments, the CTLA-4-containing protein is administered intravenously to the subject once daily for 2-5 consecutive days. In some embodiments, the CTLA-4-containing protein is administered subcutaneously to the subject once daily for 2-5 consecutive days. For example, in NAI-1540949621v1 70Atty. Docket No.14678-018-228 some embodiments, the CTLA-4-containing protein is administered subcutaneously to the subject once daily for 2, 3, 4 or 5 consecutive days. In some embodiments, the CTLA-4- containing protein is administered subcutaneously to the subject once daily for three consecutive days. In some embodiments, the IL-2 protein is administered to the subject once daily for 2-5 consecutive days. For example, in some embodiments, the IL-2 protein is administered to the subject once daily for 2, 3, 4 or 5 consecutive days. In some embodiments, the IL-2 protein is administered to the subject once daily for three consecutive days. In some embodiments, the IL- 2 protein is administered intravenously to the subject once daily for 2-5 consecutive days. In some embodiments, the IL-2 protein is administered subcutaneously to the subject once daily for 2-5 consecutive days. For example, in some embodiments, the IL-2 protein is administered subcutaneously to the subject once daily for 2, 3, 4 or 5 consecutive days. In some embodiments, the IL-2 protein is administered subcutaneously to the subject once daily for three consecutive days. In some embodiments, the CTLA-4-containing protein is abatacept. In some embodiments, the IL-2 protein is aldesleukin.

[0231] In some embodiments, according to the methods provided herein, the CTLA-4- containing protein is administered to the subject one or more times during a dosing cycle, for example 1-10 times during a dosing cycle, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times during a dosing cycle. In some embodiments, the CTLA-4-containing protein is administered to the subject as a single administration on day 1 of the dosing cycle. In some embodiments, the CTLA-4-containing protein is administered to the subject daily for a series of consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the CTLA-4-containing protein is administered to the subject daily for two consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the CTLA-4-containing protein is administered to the subject daily for three consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the CTLA-4-containing protein is administered to the subject daily for four consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the CTLA-4-containing protein is administered to the subject daily for five consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the CTLA-4-containing protein is administered to the subject for a series of non-consecutive days, beginning on day 1 of the dosing cycle, for example, administered to the subject for a series of non-consecutive days, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-consecutive days, independently separated by 1, 2, 3, 4 or 5 days, beginning on day 1 of the NAI-1540949621v1 71Atty. Docket No.14678-018-228 dosing cycle. In some embodiments, the CTLA-4-containing protein is abatacept.

[0232] In some embodiments, according to the methods provided herein, the IL-2 protein is administered to the subject one or more times during a dosing cycle, for example 1-10 times during a dosing cycle, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times during a dosing cycle. In some embodiments, the IL-2 protein is administered to the subject daily for a series of consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the IL-2 protein is administered to the subject daily for two consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the IL-2 protein is administered to the subject daily for three consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the IL-2 protein is administered to the subject daily for four consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the IL-2 protein is administered to the subject daily for five consecutive days, beginning on day 1 of the dosing cycle. In some embodiments, the IL-2 protein is administered to the subject for a series of non-consecutive days, beginning on day 1 of the dosing cycle, for example, administered to the subject for a series of non-consecutive days, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-consecutive days, independently separated by 1, 2, 3, 4 or 5 days, beginning on day 1 of the dosing cycle. In some embodiments, the IL-2 protein is aldesleukin.

[0233] In some embodiments, according to the methods provided herein, the dosing cycle is 1-6 weeks. In some embodiments, according to the methods provided herein, the dosing cycle is 2-6 weeks. In some embodiments, the dosing cycle is 1 week. In some embodiments, the dosing cycle is 2 weeks. In some embodiments, the dosing cycle is 3 weeks. In some embodiments, the dosing cycle is 4 weeks. In some embodiments, the dosing cycle is 5 weeks. In some embodiments, the dosing cycle is 6 weeks. In some embodiments, the dosing cycle is repeated 1-12 times. In some embodiments, the dosing cycle is repeated 10 times. In some embodiments, the dosing cycle is repeated 8 times. In some embodiments, the dosing cycle is repeated 6 times. In some embodiments, each repeated dosing cycle begins 10-28 days after day 1 of the previous dosing cycle. For example, in some embodiments, each repeated dosing cycle begins 14 days after day 1 of the previous dosing cycle. In some embodiments, each repeated dosing cycle begins 2-6 weeks after day 1 of the previous dosing cycle. In some embodiments, each repeated dosing cycle begins 2 weeks after day 1 of the previous dosing cycle. In some embodiments, each repeated dosing cycle begins 3 weeks after day 1 of the previous dosing cycle. In some NAI-1540949621v1 72Atty. Docket No.14678-018-228 embodiments, each repeated dosing cycle begins 4 weeks after day 1 of the previous dosing cycle. In some embodiments, each repeated dosing cycle begins 5 weeks after day 1 of the previous dosing cycle. In some embodiments, each repeated dosing cycle begins 6 weeks after day 1 of the previous dosing cycle. In some embodiments, the CTLA-4-containing protein is administered to the subject daily for three consecutive days, beginning on day 1 of a first dosing cycle. In some embodiments, the IL-2 protein is administered to the subject daily for three consecutive days, beginning on day 1 of a first dosing cycle. In some embodiments, the IL-2 protein is administered to the subject daily for three consecutive days, beginning on day 1 of the second dosing cycle. In some embodiments, the first dosing cycle is repeated 6 times, with each repeated dosing cycle beginning 14 days after day 1 of the previous dosing cycle. In some embodiments, the CTLA-4-containing protein is abatacept. In some embodiments, the IL-2 protein is aldesleukin.

[0234] In some embodiments, the CTLA4-containing protein is administered to the subject weekly. In certain embodiments, the CTLA4-containing protein is administered to the subject weekly on day 1 of each week (in other words, if the first administration of the CTLA4- containing protein is administered to the subject on day 1, subsequent administrations of the CTLA4-containing protein occur at day 8, day 15, day 22 etc.). In some embodiments, the CTLA-4-containing protein is abatacept.

[0235] In specific embodiments, the CTLA4-containing protein is administered to the subject weekly and the IL-2 protein is administered to the subject every two weeks. For example, in certain embodiments, the CTLA4-containing protein is administered to the subject weekly beginning with week 1, and the IL-2 protein is administered to the subject on week 2, week 4, week 6 etc. For example, in certain embodiments, the CTLA4-containing protein is administered to the subject weekly beginning with week 1, and the IL-2 protein is administered to the subject on week 3, week 5, week 7 etc. In some embodiments, the CTLA-4-containing protein is abatacept. In some embodiments, the IL-2 protein is aldesleukin.

[0236] In certain embodiments, the CTLA4-containing protein is administered to the subject weekly on day 1 of each week (in other words, if the first administration of the CTLA4- containing protein is administered to the subject on day 1, subsequent administrations of the CTLA4-containing protein occur at day 8, day 15, day 22 etc.) and the IL-2 protein is administered to the subject on, or beginning on, day 1 of every other week, beginning with week NAI-1540949621v1 73Atty. Docket No.14678-018-228 2 (in other words, day 8, day 22, day 36 etc). In certain embodiments, IL-2 protein is administered to the subject on day 1 of an IL-2 protein administration week for a series of consecutive days, e.g., daily for two consecutive days, daily for three consecutive days, daily for four consecutive days or daily for five consecutive days. In certain embodiments, IL-2 protein is administered to the subject on day 1 of an IL-2 protein administration week for a series of non- consecutive days, such as 2, 3, 4, 5, 6 or 7 non-consecutive days, independently separated by 1, 2, 3, 4 or 5 days. In some embodiments, the CTLA-4-containing protein is abatacept. In some embodiments, the IL-2 protein is aldesleukin.

[0237] In certain embodiments, the CTLA4-containing protein is administered to the subject weekly on day 1 of week 1 (in other words, if the first administration of the CTLA4- containing protein is administered to the subject on day 1, subsequent administrations of the CTLA4-containing protein occur at day 8, day 15, day 22 etc.) and the IL-2 protein is administered to the subject on, or beginning on, day 1 of every other week, beginning with week 3 (in other words, day 15, day 29, day 53, etc). In certain embodiments, IL-2 protein is administered to the subject on day 1 of an IL-2 protein administration week for a series of consecutive days, e.g., daily for two consecutive days, daily for three consecutive days, daily for four consecutive days or daily for five consecutive days. In certain embodiments, IL-2 protein is administered to the subject on day 1 of an IL-2 protein administration week for a series of non- consecutive days, such as 2, 3, 4, 5, 6 or 7 non-consecutive days, independently separated by 1, 2, 3, 4 or 5 days. In some embodiments, the CTLA-4-containing protein is abatacept. In some embodiments, the IL-2 protein is aldesleukin.

[0238] In some embodiments, according to the methods provided herein, the CTLA4- containing protein is administered to the subject weekly, e.g., as described in any of the embodiments presented herein in an amount in the range of 20-50 mg per week. In certain embodiments, according to the methods provided herein, the CTLA4-containing protein is administered to the subject weekly, e.g., as described in any of the embodiments presented herein in an amount in the range of 20 mg per week. In certain embodiments, according to the methods provided herein, the CTLA4-containing protein is administered to the subject weekly, e.g., as described in any of the embodiments presented herein in an amount in the range of 25 mg per week. In certain embodiments, according to the methods provided herein, the CTLA4- containing protein is administered to the subject weekly, e.g., as described in any of the NAI-1540949621v1 74Atty. Docket No.14678-018-228 embodiments presented herein in an amount in the range of 30 mg per week. In certain embodiments, according to the methods provided herein, the CTLA4-containing protein is administered to the subject weekly, e.g., as described in any of the embodiments presented herein in an amount in the range of 35 mg per week. In certain embodiments, according to the methods provided herein, the CTLA4-containing protein is administered to the subject weekly, e.g., as described in any of the embodiments presented herein in an amount in the range of 40 mg per week. In certain embodiments, according to the methods provided herein, the CTLA4- containing protein is administered to the subject weekly, e.g., as described in any of the embodiments presented herein in an amount in the range of 45 mg per week. In certain embodiments, according to the methods provided herein, the CTLA4-containing protein is administered to the subject weekly, e.g., as described in any of the embodiments presented herein in an amount in the range of 50 mg per week. In certain embodiments, the CTLA4-containing protein is abatacept. In some embodiments, the IL-2 protein, e.g., aldesleukin, administered in conjunction with methods comprising weekly administration of the CTLA4-containing protein, e.g., abatacept, is administered in an amount in the range of 10,000-3,000,000 units. In some such embodiments, the IL-2 protein, e.g., aldesleukin, is administered in an amount in the range of 500,000-3,000,000 units. In some such embodiments, the IL-2 protein, e.g., aldesleukin, is administered in an amount in the range of 500,000-2,000,000 units. In some embodiments, the IL-2 protein, e.g., aldesleukin, is administered in an amount of 1,000,000 units. In some such embodiments, the IL-2 protein, e.g., aldesleukin, is administered in an amount in the range of about 25-1000 mg, about 25-750 mg, about 25-500 mg, about 25-250 mg, about 25-100 mg, or about 25-50 mg. In some embodiments, the IL-2 protein, e.g., aldesleukin, is administered in an amount of 25 mg, about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 750 mg, or about 1000 mg. In some embodiments, the IL-2 protein, e.g., aldesleukin can be administered at a low dose (see Section 7.4.2(b)). In some embodiments, the IL-2 protein, e.g., aldesleukin can be administered according to a regimen of low dose IL-2 administration as described in Section 7.4.2(b)).

[0239] In certain embodiments, the CTLA-4-containing protein is administered by injection or infusion. In particular embodiments, the CTLA-4-containing protein is administered subcutaneously. In particular embodiments, the CTLA-4-containing protein is administered intravenously. In certain embodiments, the IL-2 protein is administered by injection or infusion. NAI-1540949621v1 75Atty. Docket No.14678-018-228 In particular embodiments, the IL-2 protein is administered subcutaneously. In particular embodiments, the IL-2 protein is administered intravenously. In certain embodiments, the CTLA-4-containing protein and the IL-2 protein are administered by injection or infusion. In particular embodiments, the CTLA-4-containing protein and the IL-2 protein are administered subcutaneously. In particular embodiments, the CTLA-4-containing protein and the IL-2 protein are administered intravenously. 7.5 Palliative Care

[0240] In certain embodiments, a therapeutic regimen used herein is palliative care. In certain embodiments, a therapeutic regimen used herein comprises palliative care.

[0241] In some embodiments, palliative care can be a therapeutic regimen for treating ALS as disclosed herein. In some embodiments, palliative care comprises management of one or more symptoms of ALS. One of skill in the art can appreciate that the methods of managing the one or more symptoms of ALS are known in the art and a skilled artisan can select the most suitable method for use in administering palliative care as disclosed herein. In some embodiments, palliative care can comprise the management of generalized fatigue and / or muscle specific fatigue in the ALS subject. In some embodiments, palliative care can comprise the management of impaired mobility in the ALS subject. In some embodiments, palliative care can comprise the management of pain in the ALS subject. In some embodiments, palliative care can comprise the management of respiratory insufficiency in the ALS subject. In some embodiments, palliative care can comprise the management of secretions and airway clearance in the ALS subject. In some embodiments, palliative care can comprise the management of dysphagia in the ALS subject. In some embodiments, palliative care can comprise the management of dysarthria in the ALS subject. In some embodiments, palliative care can comprise the management of laryngospasms in the ALS subject. In some embodiments, palliative care can comprise the management of pseudobulbar affect in the ALS subject. In some embodiments, palliative care can comprise the management of cognitive decline in the ALS subject.

[0242] In certain embodiments, palliative care for use herein comprises administering an anti-inflammatory agent. Non-limiting examples of anti-inflammatory agents include aspirin, non-aspirin nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, and the like. In certain embodiments, the first and / or second therapeutic regimen comprises administering an NAI-1540949621v1 76Atty. Docket No.14678-018-228 antioxidant agent. Non-limiting examples of anti-antioxidant agents include riluzole, edaravone, masitinib, (R)-troloxamide quinone, phytochemicals, and the like. 7.6 Kits

[0243] Also provided in the present disclosure are kits for determining the level of 4-HNE as disclosed herein. In certain embodiments, the kits described herein can be used to predict the survival time in a subject, determine impact of an ALS therapy on survival of a subject, and / or to predict the rate of progression of ALS to an endpoint in a subject.

[0244] In certain embodiments, kits described herein can be used for determining the level of 4-HNE in a subject, wherein the kit comprises an antigen-binding molecule specific for 4- HNE which allows for measuring the level of 4-HNE in a biological sample.

[0245] In certain embodiments, the kits disclosed herein can also comprise suitable detection agents, including for example, conjugates and substrates to facilitate detection (e.g., antibodies labelled with streptavidin, biotin, horseradish peroxidase, etc.). In some embodiments, kits disclosed herein comprise at least one reagent for detecting formation of a 4-HNE-antibody complex. In some other embodiments, kits disclosed herein can also feature various devices and additional reagents and / or buffers, and / or printed instructional materials for using the kit to detect and / or quantify levels of 4-HNE. The kit reagents described herein, which may be optionally associated with detectable labels, can be presented in the format of a plurality of beads, a multiwall plate, a microarray, slide, microfluidics card, or a chip adapted for use with the techniques described herein for the measurement of 4-HNE levels in a sample. In some embodiments, a kit can include a control and / or a reference (e.g., a reference antibody and / or a reference sample from a healthy subject).

[0246] In certain embodiments, kits disclosed herein can comprise solid supports and / or compositions for use in methods of determining 4-HNE in a biological sample (e.g., serum), comprising one or more antigen-binding molecules (e.g., an antibody or antigen-binding fragment thereof) that bind to 4-HNE as described above and herein. In some embodiments, the solid supports and / or compositions comprise an antigen-binding molecule specific for 4-HNE. Examples of solid supports for use in the kits disclosed herein include, but are not limited to, multiwell plates, slides, beads, and chips. As described above, these solid supports can be used in multiplex immunoassays (e.g., ELISA) to determine the level of 4-HNE in a biological sample (e.g., serum, plasma). NAI-1540949621v1 77Atty. Docket No.14678-018-228

[0247] In particular embodiments, a kit as described herein comprises one or more components for assessing whether the 4-HNE level in a sample is lower than, equal to or greater than a 4-HNE reference range. For example, in certain embodiments, kits described herein can be colorimetric kits used for determining the level of 4-HNE in a subject, wherein the kit comprises at least 2 reference samples for indicating the reference range of 4-HNE. In some embodiments, a sample tested in the kit disclosed herein emits a colorimetric readout that is the same colorimetric readout of the reference samples, indicating that the biological sample is equal to the reference range of 4-HNE. In some embodiments, a sample tested in the kit disclosed herein emits a colorimetric readout that is not the same colorimetric readout of the reference samples, indicating that the biological sample is not equal to the reference range of 4-HNE. In certain embodiments, colorimetric assay kits described herein further comprise at least 2 reference samples for indicating the reference range of 4-HNE, a “long-term survivor” reference sample for indicating 4-HNE expression levels below the reference range of 4-HNE, and a “short-term survivor” reference sample for indicating 4-HNE expression levels above the reference range of 4-HNE. In some embodiments, the reference samples, the long-term survivor reference sample, and the short-term survivor reference sample have different colorimetric readouts. In some embodiments, a sample tested in the kit disclosed herein emits a colorimetric readout that is not the same colorimetric readout of the reference samples but is the same as the long-term survivor reference sample, indicating 4-HNE expression levels are below the reference range of 4-HNE. In some embodiments, a sample tested in the kit disclosed herein emits a colorimetric readout that is not the same colorimetric readout of the reference samples but is the same as the short-term survivor reference sample, indicating 4-HNE expression levels are above the reference range of 4-HNE. 7.7 Patient Populations

[0248] ALS, also known as Lou Gehrig disease, is a fatal disorder characterized by progressive skeletal muscle weakness and wasting or atrophy (i.e., amyotrophy), spasticity, and fasciculations as a result of degeneration of the UMNs and LMNs, culminating in respiratory paralysis. In general, ALS is classified as sporadic, familial, or Western Pacific ALS with or without Parkinsonism-dementia complex (ALS / PDC). Most ALS cases are sporadic, and only 5- 10% of cases are considered to be familial. Mutations in the C9orf72 gene are responsible for 30-40% of familial ALS cases in the United States and Europe. Worldwide, approximately 20% NAI-1540949621v1 78Atty. Docket No.14678-018-228 of cases of familial ALS are due to a mutation in the Cu / Zn superoxide dismutase-1 gene (SOD1).

[0249] Described herein are methods for predicting ALS disease survival and / or ALS disease progression in a subject. The term “subject” and “patient” are used interchangeably throughout the present disclosure. Subjects for whom the methods provided herein can be used can be diagnosed and selected for based on assessing diagnostic criteria associated with ALS according to the methods described in Section 7.8.1. In certain embodiments, subjects for whom the methods provided herein can be used may have a particular stage of ALS at the time of treatment. Methods of determining disease stage are detailed in Section 7.8.2 below.

[0250] In certain embodiments, a subject to the methods disclosed herein is a mammal. In some embodiments, the subject is a human subject. In some embodiments, the subject is an adult human subject. In some embodiments, the subject is a female human subject. In some embodiments, the subject is a male human subject. In some embodiments, the human subject is between about 18 to about 90 years of age. In some embodiments, the human subject is over 65, over 70, over 75, over 80, over 85, or over 90 years of age.

[0251] In certain embodiments, a subject to the methods disclosed herein has been diagnosed has having ALS according to Gold Coast criteria (see Section 7.8.1(c)).

[0252] In certain embodiments, subjects to the methods disclosed herein can include subjects diagnosed as having possible, laboratory supported probable, probable, or definite ALS according to revised El Escorial criteria (see Section 7.8.1(a)). In some embodiments, a subject to the methods disclosed herein can be a subject diagnosed as having possible ALS. In some embodiments, a subject to the methods disclosed herein can be a subject diagnosed as having laboratory supported probable ALS. In some embodiments, a subject to the methods disclosed herein can be a subject diagnosed as having probable ALS. In some embodiments, a subject to the methods disclosed herein can be a subject diagnosed as having definite ALS.

[0253] In certain embodiments, subjects to the methods disclosed herein can include subjects diagnosed as having clinically definite, clinically probable, or clinically possible ALS according to Awaji Shima criteria (See Section 7.8.1(b)) In some embodiments, a subject to the methods disclosed herein can be a subject diagnosed as having clinically definite ALS. In some embodiments, a subject to the methods disclosed herein can be a subject diagnosed as having clinically probable ALS. In some embodiments, a subject to the methods disclosed herein can be NAI-1540949621v1 79Atty. Docket No.14678-018-228 a subject diagnosed as having clinically possible ALS.

[0254] In some embodiments, the subject is a human subject recently diagnosed with ALS (e.g., diagnosed as having first symptoms within 3 months to 3 years from diagnosis). In some embodiments, the subject is a human subject diagnosed as having first symptoms within 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months from the start of administration of one or more ALS therapies disclosed herein.

[0255] In some embodiments, the human subject is a naïve subject (i.e., a subject that has not been treated for ALS). In some embodiments, the human subject has been previously treated with an agent that is used for treating ALS. In some embodiments, the human subject was not responsive to previous ALS treatment(s). In some embodiments, the subject is unresponsive to at least one treatment for ALS. In some embodiments, the subject is unresponsive to several treatments of ALS. In some embodiments, the subject has sporadic ALS. In some embodiments, the subject has familial ALS. In some embodiments, the subject has a genetic mutation associated with ALS (e.g., mutation in C9orf72, SOD1, TARDBP and / or FUS). In some embodiments, the subject does not have another acute or chronic disorder of the immune system (e.g., only ALS). In some embodiments, the subject does not have another immune related disorder (e.g., only ALS). In some embodiments, the subject does not have another chronic or acute inflammatory disorder of the immune system (e.g., only ALS). In some embodiments, the subject does not have another disease or condition besides ALS. In some embodiments, the subject has another acute or chronic disorder of the immune system (e.g., in addition to ALS). In some embodiments, the subject has another immune related disorder (e.g., in addition to ALS). In some embodiments, the subject has another chronic or acute inflammatory disorder of the immune system (e.g., in addition to ALS). In some embodiments, the subject has another disease or condition in addition to ALS.

[0256] In certain embodiments, patients for whom the methods provided herein can be used include ALS patients at various disease stages (see Section 7.8.2). In some embodiments, ALS patients for whom the methods provided herein can be used can be staged according to the King’s system of staging. In some embodiments, ALS patients for whom the methods provided NAI-1540949621v1 80Atty. Docket No.14678-018-228 herein can be used are stage 1 ALS patients, stage 2 ALS patients, stage 3 ALS patients, and / or stage 4 ALS patients according to the King’s system of staging. In some embodiments, an ALS patient for whom the methods provided herein can be used is a stage 1 ALS patient according to the King’s system of staging. In some embodiments, an ALS patient for whom the methods provided herein can be used is a stage 2 ALS patient according to the King’s system of staging. In some embodiments, an ALS patient for whom the methods provided herein can be used is a stage 3 ALS patient according to the King’s system of staging. In some embodiments, an ALS patient for whom the methods provided herein can be used is a stage 4 ALS patient according to the King’s system of staging. In some preferred embodiments, an ALS patient for whom the methods provided herein can be used is a stage 2 ALS patient according to the King’s system of staging. In some other preferred embodiments, an ALS patient for whom the methods provided herein can be used is a stage 1 ALS patient according to the King’s system of staging.

[0257] In some embodiments, ALS patients for whom the methods provided herein can be used can be staged according to the MiToS system of staging. In some embodiments, ALS patients for whom the methods provided herein can be used are stage 1 ALS patients, stage 2 ALS patients, stage 3 ALS patients, and / or stage 4 ALS patients according to the MiToS system of staging. In some embodiments, an ALS patient for whom the methods provided herein can be used is a stage 0 ALS patient according to the MiToS system of staging, wherein the patient reports one or more physiological symptoms of ALS. In some embodiments, an ALS patient for whom the methods provided herein can be used is a stage 1 ALS patient according to the MiToS system of staging. In some embodiments, an ALS patient for whom the methods provided herein can be used is a stage 2 ALS patient according to the MiToS system of staging. In some embodiments, an ALS patient for whom the methods provided herein can be used is a stage 3 ALS patient according to the MiToS system of staging. In some embodiments, an ALS patient for whom the methods provided herein can be used is a stage 4 ALS patient according to the MiToS system of staging. In some preferred embodiments, an ALS patient for whom the methods provided herein can be used is a stage 2 ALS patient according to the MiToS system of staging. In some other preferred embodiments, an ALS patient for whom the methods provided herein can be used is a stage 1 ALS patient according to the MiToS system of staging. 7.8 Diagnostic Criteria and Disease Staging

[0258] Provided herein are various methods for selecting patients for treatment with the NAI-1540949621v1 81Atty. Docket No.14678-018-228 methods disclosed herein. Also provided herein are methods of selecting patients with various stages of ALS to be treated with the methods disclosed herein. Methods of diagnosing subjects to be treated with a method disclosed herein can include assessing diagnostic criteria (Section 7.8.1) in the subject. Methods of determining disease stage are detailed in Section 7.8.2 below. 7.8.1 Diagnostic Criteria

[0259] Various diagnostic criteria are available for diagnosing and / or predicting the onset of ALS. Exemplary diagnostic criteria for ALS include El Escorial criteria, Awaji Shima criteria, and Gold Coast criteria. (a) El Escorial Criteria

[0260] An ALS diagnosis may be carried out according to revised El Escorial criteria as described in Ludolph A et al., Amyotroph Lateral Scler Frontotemporal Degener.2015;16(5- 6):291-2. According to such analysis, a subject’s upper motor neuron (UMN) and lower motor neuron (LMN) impairment is assessed in bulbar, cervical, thoracic, and lumbosacral regions of the body. UMN assessment includes analysis of spasticity and hyper-reflexivity. LMN assessment includes assessment of weakness, atrophy, and fasciculation. Subjects with signs of UMN and LMN impairment in at least three regions tested are diagnosed as having “definite” ALS. Subjects with signs of UMN and LMN impairment in at least two regions tested are diagnosed as having “probable” ALS. Subjects with UMN and LMN signs in one region with evidence by electromyography (EMG) of LMN involvement in another region are characterized as having “laboratory supported probable” ALS. Subjects with signs of UMN and LMN impairment in one region tested or have UMN impairment alone in two or more regions are characterized as having “possible” ALS. Subjects with signs of LMN impairment alone in two or more regions tested are characterized as having “suspected” ALS. (b) Awaji Shima criteria

[0261] ALS may be diagnosed according to Awaji Shima criteria as described in deCarvalho M et al., Clin Neurophysiol.2008;119(3):497-503. Awaji Shima criteria integrate electrophysiological criteria with clinical examination findings where the electrophysiological evidence is considered of equal weight to the clinical exam findings of lower motor neuron abnormality. In some examples, Awaji Shima criteria and El Escorial criteria can be used in combination to diagnose a subject as having ALS. Electrophysiological criteria are assessed by EMG, a technique known in the art that detects electrical activity in muscles. EMG is useful in NAI-1540949621v1 82Atty. Docket No.14678-018-228 detecting the findings of acute denervation (fibrillation and positive sharp waves), chronic denervation (long-duration, complex motor unit action potentials [MUAP]), and chronic reinnervation (large amplitude MUAP).

[0262] To be indicative of ALS according to Awaji Shima criteria, an EMG generally shows signs of acute or chronic denervation in at least three spinal levels (bulbar, cervical, thoracic, and / or lumbosacral). If three spinal levels are not abnormal, acute or chronic denervation needs to be evident in three extremities with the involvement of at least two muscles supplied by two different roots and two different nerves in each extremity. According to Awaji Shima criteria, “clinically definite ALS” is defined by clinical or electrophysiological evidence by the presence of LMN as well as UMN signs in the bulbar region and at least 2 spinal regions or the presence of LMN and UMN signs in 3 spinal regions. “Clinically probable ALS” is defined by clinical or electrophysiological evidence of LMN and UMN signs in at least 2 regions with some UMN signs necessarily rostral to (above) the LMN signs. “Clinically possible ALS” is defined when clinical or electrophysiological signs of UMN and LMN dysfunction are found in only 1 region or UMN signs are found alone in 2 regions or LMN signs are found rostral to UMN signs. (c) Gold Coast Criteria

[0263] ALS may be diagnosed in a subject according to Gold Coast criteria as described in Shefner JM et al., Clin Neurophysiol 2020 Aug;131(8):1975-1978. According to Gold Coast criteria, a subject may be diagnosed as having ALS if: 1) progressive motor impairment was documented by history or repeated clinical assessment, preceded by normal motor function; 2) upper and lower motor neuron dysfunction is present in at least one body region with either (i) upper and lower motor neuron dysfunction noted in the same body region if only one region is involved or (ii) lower motor neuron dysfunction in at least two body regions; and 3) other disease processes are excluded. Upper motor neuron dysfunction implies the presence of at least one of the following: increased deep tendon reflexes, including the presence of a reflex in a clinically weak and wasted muscle, or spread to adjacent muscles; or the presence of pathological reflexes, including Hoffman sign, Babinski sign, crossed adductor reflex, or snout reflex. Lower motor neuron dysfunction in a given muscle requires either: (1) clinical examination evidence of muscle weakness and muscle wasting; or (2) EMG abnormalities that generally include both (i) evidence of chronic neurogenic change, defined by large motor unit potentials of increased duration and / or increased amplitude (with polyphasia) and motor unit instability regarded as NAI-1540949621v1 83Atty. Docket No.14678-018-228 supportive but not obligatory evidence, and (ii) evidence of ongoing denervation, including fibrillation potentials or positive sharp waves, or fasciculation potentials. 7.8.2 Disease Staging

[0264] Various staging systems are available for measuring the severity and / or rate of progression of ALS. Exemplary staging systems include the King’s Clinical Staging system and the Milano–Torino Staging (MiToS) system. The MiToS system generally has a higher resolution in late disease, when functional involvement has developed, and the King’s system generally has a higher resolution in early disease to mid-disease, when clinical or disease burden is increasing.

[0265] The King’s system uses five stages, from 1 to 5 and is based on disease burden as measured by clinical involvement and significant feeding or respiratory failure, with stage 1 being symptom onset and stage 5 being death. ALS progression is defined by loss of independence in four key domains on the ALS Functional Rating Scale (ALSFRS): swallowing, walking / self-care, communicating and breathing. The King’s system defines the stages as follows: stage 0, functional involvement but no loss of independence on any domain; stages 1-4, number of domains in which independence was lost; and stage 5, death.

[0266] The MiToS system involves six stages (0–5) based on functional ability assessed with the ALSFRS-R; stage 0 is defined as normal function and stage 5 is death. The MiToS stage at diagnosis correlates with survival at 18 months. Stage 1: symptom onset (involvement of first region); Stage 2A: diagnosis; Stage 2B: involvement of second region; Stage 3: involvement of third region; Stage 4A: need for gastrostomy; and Stage 4B: need for non-invasive ventilation. 8. EXAMPLES

[0267] The following is a description of various methods and materials used in the studies. They are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental NAI-1540949621v1 84Atty. Docket No.14678-018-228 errors and deviations should be accounted for. 8.1 Example 1. Correlative and predictive values of 4-HNE and ox-LDL sera expression levels in ALS patients.

[0268] Study Subjects. Fifty adult patients with ALS meeting El Escorial criteria for possible, probable, laboratory-supported probable, or definite ALS were randomly chosen from a data bank of patients that died between January 2018 and December 2022. The mean age of the fifty patients was 60.88 ± 9.69 years. Thirty-one of the patients were men (62%) and 19 were women (38%); 44 were Caucasian (88%), 4 were Black (8%), and 2 were Hispanic (4%).

[0269] Of the fifty adult patients with ALS, thirty patients had limb onset (60%), 17 patients had bulbar onset (34%), and 3 patients had other (6%). The mean time from disease onset to diagnosis was 2.15 ± 1.69 years. All patients were diagnosed with sporadic ALS.

[0270] ALSFRS-R scores were determined according to the methods described in Cedarbaum JM, J Neurol Sci 1999; 169: 13-21, the disclosure of which is incorporated herein in its entirety. ALSFRS-R scores were determined prior to initiation of blood sample collection. First blood draws were at diagnosis. Serial blood samples and clinical information were obtained on average every three to four months at clinic visits from the 50 patients with ALS, who accepted to donate further blood samples after first blood draw. Clinical information determined and recorded for each sample time included an Amyotrophic Lateral Sclerosis Functional Rating Scale (ALS-FRS) score, an AALS scores, respiratory status, and presence of feeding tube (Yes / No). All patients received standard care for ALS when appropriate. No patient was subjected to any investigational ALS therapy.

[0271] For comparison, fifty-three age matched control individuals (“healthy controls”) were selected that were similar in age (61.6 ± 6.49 years). Thirty-two were men (60%) and 21 were women (40%); 90.0% were Caucasian, none were Hispanic, none were Black, and 10% were Asian.

[0272] Assessment of 4-HNE and ox-LDL Serum Concentrations in Subjects. Serum was collected from blood samples drawn from the study subjects according to standard procedures in the art. The resulting serum was subjected to an enzyme-linked immunosorbent assay (ELISA) to assess 4-HNE and ox-LDL concentrations in the serum of subjects with ALS and healthy controls (HC) according to manufacturer’s (Cell Biolabs, Inc.) instructions. For 4-HNE, the Cell Biolabs, Inc. ELISA kit had an assay range from 1.56 µg / ml to 100 µg / ml. For ox-LDL, the Cell NAI-1540949621v1 85Atty. Docket No.14678-018-228 Biolabs, Inc. ELISA kit had an assay range from 1.00 mU / L to 25.0 mU / L, which was extended to a range of 6.56 U / L to 164 U / L when multiplied by a dilution factor (6561:1), with a lower limit of detection (LLOD) ≤ 0.5 mU / L.

[0273] Statistics. Comparisons were performed using an ANOVA for more than two groups or Student’s t-test for two groups. The ANOVA is presented with the degrees of freedom, F value, and p value. The Student’s t-test is presented with a p value. Correlations were done using Spearman Rank Order in SigmaStat software and presented with a r (rho) and p values. The Spearman’s correlation coefficient is r and has a range between -1 and 1 where: -1 indicates a strong negative correlation between two variables; 0 indicates that there is no association between the two variables; and 1 indicates a strong positive correlation between two variables.

[0274] Results. To determine if the rate of ALS disease progression correlates with survival, the rate of ALS disease progression for each of the 50 ALS patients was determined. The rate of disease progression for each of the 50 ALS patients studied longitudinally was correlated with the number of months the patient survived after the date of onset of the first ALS symptom (FIG. 1A), after the date of receiving an ALS diagnosis (FIG.1B), and after the date of collecting the last sample ALS-FRS score before death (FIG.1C). For these 50 ALS patients, the time between collecting the last sample ALS-FRS score and death varied from 1 month to over 100 months. The rate of disease progression for each of the 50 ALS patients studied longitudinally was also correlated with the duration of time (months) between the date of symptom onset to the date of diagnosis (FIG.1D). The data show that the rate of disease progression correlates with survival from the date of onset to death (r = -0.663), from the date of diagnosis to death (r = -0.723), as well as from the last sample collection date to death (r = -0.499), and, to a lesser extent, from the duration of time between symptom onset to ALS diagnosis (r = -0.383).

[0275] Relationship between ALS and 4-HNE expression. To determine if 4-HNE expression correlates with survival, 4-HNE serum concentration was measured in each serial serum sample collected from the 50 ALS patients using ELISA as described above. For a single patient, the 4- HNE serum concentration measurement from every serial sample was averaged to provide a mean 4-HNE expression level for that patient. Mean 4-HNE expression levels in the serum of each of the 50 ALS patients studied longitudinally was correlated to their survival time from the date of onset of disease to the date of death (FIG.2A) and to their survival time from the date of diagnosis of ALS disease to the date of death (FIG.2B) using the Spearman correlation method NAI-1540949621v1 86Atty. Docket No.14678-018-228 which resulted in r values of -0.738 and -0.696, respectively. The 4-HNE expression level in the last serum sample collected from each of the 50 ALS patients prior to death was correlated to duration of the time between the date that the last sample was collected and the date of death using the Spearman correlation method, resulting in an r value of -0.712 (FIG.2C). For these 50 ALS patients, the time between collecting the last serum sample and death varied from 1 month to over 100 months. These data demonstrated strong correlations among mean 4-HNE expression levels in the serum of each of the 50 patients with ALS studied longitudinally and their survival times from 1) date of onset of disease to date of death, 2) date of diagnosis of disease to date of death, and 3) from the date of last sample collection to date of death.

[0276] The 4-HNE expression level in the serum sample collected at the time of diagnosis from each of the 50 ALS patients was correlated to survival time from the date of onset of ALS disease to the date of death using the Spearman correlation method, resulting in an r value of -0.602 (FIG.3A). The 4-HNE expression level in the serum sample collected at the time of diagnosis from each of the 50 ALS patients was also correlated to survival time from the date of ALS disease diagnosis to the date of death using the Spearman correlation method, resulting in an r value of -0.435 (FIG.3B). Additionally, the 4-HNE expression level in the serum sample collected at the time of diagnosis from each of the 50 ALS patients was correlated to the duration of time between the date of onset of disease and the date of ALS disease diagnosis using the Spearman correlation method, resulting in an r value of -0.500 (FIG.3C). These data demonstrate that there were strong correlations among 4-HNE expression levels at the time of diagnosis in the serum of each of the 50 ALS patients studied longitudinally and their survival times from 1) the date of onset of disease to the date of death, 2) the date of diagnosis of disease to the date of death, and 3) from the date of onset to the date of diagnosis.

[0277] The 4-HNE expression level in the serum samples collected at the time of diagnosis was determined for male ALS patients (n=31) and female ALS patients (n=19). The 4-HNE expression at the time of diagnosis in male ALS patients and female ALS patients was correlated to survival time from the date of onset of disease to the date of death using the Spearman correlation method, resulting in r values of -0.567 (FIG.4A) and -0.453 (FIG.4B), respectively. The 4-HNE expression at the time of diagnosis in male ALS patients and female ALS patients was also correlated to survival time from the date of ALS disease diagnosis to the date of death using the Spearman correlation method, resulting in r values of -0.441 (FIG.4C) and -0.220 NAI-1540949621v1 87Atty. Docket No.14678-018-228 (FIG.4D), respectively.

[0278] To determine if 4-HNE serum expression levels correlate with disease progression, the rate of disease progression for each of the 50 ALS patients was determined as described above. Mean 4-HNE expression levels in the serum of each of the 50 ALS patients studied longitudinally was correlated to the rate of disease progression determined for each of the 50 ALS patients using the Spearman correlation method which resulted in an r value of 0.432 (FIG. 5A). The 4-HNE expression level in the serum sample collected at the time of diagnosis from each of the 50 ALS patients was correlated to the rate of disease progression determined for each of the 50 ALS patients using the Spearman correlation method, resulting in an r value of 0.318 (FIG.5B). The 4-HNE expression level in the last serum sample collected from each of the 50 ALS patients prior to death was correlated to the rate of disease progression determined for each of the 50 ALS patients using the Spearman correlation method, resulting in an r value of 0.375 (FIG.5C). FIGS.5A-5C show the significant, moderate correlative value of 1) the mean 4- HNE serum level to the rate of ALS disease progression, 2) the 4-HNE serum level measured at the time of diagnosis to the rate of disease progression, and 3) the 4-HNE serum expression level measured prior to death to the rate of disease progression.

[0279] To determine if the rate of change of 4-HNE serum expression levels correlates with disease progression, the rate of disease progression for each of the 50 ALS patients was determined as described above. In FIG.6A, the rate of change in 4-HNE levels was determined from the concentration change between a single patient sample taken on the first day of the month and a single patient sample taken on the last day of the month. The rate of change in 4- HNE levels per month was then averaged for each individual ALS patient and correlated to the rate of disease progression (i.e., the average change in ALS-FRS score per month). Using the Spearman correlation method, the correlative data presented in FIG.6A demonstrate an r value of 0.421. In FIG.6B, the rate of change in mean 4-HNE levels was determined as an average of the measured change in 4-HNE levels between two serial sample collections from the course of all serial samples taken from a patient between the first day of the month to the last day of the month. The rate of change in mean 4-HNE levels per month was averaged for each individual ALS patient and then correlated to the rate of disease progression. Using the Spearman correlation method, the correlative data presented in FIG.6B demonstrate an r value of 0.637. These data show that the rate of change in 4-HNE expression levels over the course of disease in NAI-1540949621v1 88Atty. Docket No.14678-018-228 the 50 ALS patients studied longitudinally correlate with the patient’s rate of disease progression. This was observed for both the change in 4-HNE expression levels from 1) the first 4-HNE sample assay to the last 4-HNE sample assay per month and 2) the mean 4-HNE expression level over the course of sample assays per month.

[0280] The rate of change of 4-HNE serum expression levels was also correlated with survival for each of the 50 ALS patients. For these studies, the rate of change in 4-HNE levels was determined from the difference in 4-HNE amounts between a single patient sample taken on the first day of the diagnosis and a single patient sample taken prior to death (i.e., the last sample collected before death) divided by the number of months between the two sample collections. The change in 4-HNE levels from the first 4-HNE expression amount measured at diagnosis to the 4-HNE expression amount measured at the last serial sample collection before death for each individual ALS patient was correlated to the age of that individual patient’s age at the date of onset to their age at the date of death using the Spearman correlation method, resulting in an r value of -0.539 (FIG.7A). The change in 4-HNE levels from the first 4-HNE expression amount measured at diagnosis to the 4-HNE expression amount measured at the last serial sample collection before death for each individual ALS patient was correlated to the age of that individual patient’s age at the date of ALS diagnosis to their age at the date of death using the Spearman correlation method, resulting in an r value of -0.693 (FIG.7B). Similar to the correlative value of 4-HNE serum expression levels with rates of disease progression shown in FIGS.6A-6B, these data demonstrated that the change in 4-HNE serum expression levels over the course of the patient’s disease – from first 4-HNE expression levels at diagnosis to 4-HNE expression levels at last sample collection – correlated with survival from 1) age at ALS diagnosis to the age of death and 2) age at onset of ALS disease and age at death.

[0281] Relationship between ALS and ox-LDL expression. To determine whether expression of another biomarker, ox-LDL, also correlated with survival in ALS, the 4-HNE and ox-LDL serum expression in each of the serial blood samples collected from the 50 ALS patients and the 53 healthy control subjects was measured by ELISA as described above. Without wishing to be bound by any particular theory, 4-HNE and ox-LDL can both be a reflection of oxidative stress in a sample. However, 4-HNE and ox-LDL are produced by different cells. 4-HNE is likely derived from macrophages / microglia or other myeloid cells, whereas ox-LDL is produced by a wide range of cell types. NAI-1540949621v1 89Atty. Docket No.14678-018-228

[0282] For a single patient, the 4-HNE and ox-LDL serum concentrations from each serial sample was averaged to provide one mean value of 4-HNE and ox-LDL expression level for that patient. In FIG.8A, the mean 4-HNE expression level for each of the 50 ALS patients was compared to the mean 4-HNE expression level for each of the 53 healthy control subjects. In FIG.8B, the mean ox-LDL expression level for each of the 50 ALS patients was compared to the mean ox-LDL expression level for each of the 53 healthy control subjects. These data demonstrate that the mean 4-HNE and mean ox-LDL expression level in the serum of each patient with ALS was significantly elevated compared with the mean expression level of 4-HNE and ox-LDL in the serum of age-matched healthy controls.

[0283] To determine if 4-HNE serum expression correlated with ox-LDL serum expression, the serum expression levels were measured in every serial sample collected from all 50 ALS patients for a total of 2984-HNE and ox-LDL matched data points. Correlation of the matched data points determined according to the Spearman correlation method resulted in an r value of 0.221 (FIG.9). These data demonstrate that there was a weak correlation between serum

[0284] Mean ox-LDL expression levels in the serum of each of the 50 ALS patients studied longitudinally was correlated to their survival time from the date of onset of disease to the date of death (FIG.10A) and their survival time from the date of diagnosis of ALS disease to the date of death (FIG.10B) using the Spearman correlation method which resulted in r values of -0.066 and -0.176, respectively. The ox-LDL expression level in the last serum sample collected from each of the 50 ALS patients prior to death was correlated to the duration of time between the date of the last sample collection and the date of death using the Spearman correlation method, resulting in an r value of -0.251 (FIG.10C). The ox-LDL expression level in the serum sample collected at the time of diagnosis from each of the 50 ALS patients was correlated to survival time from the date of onset of disease to the date of death using the Spearman correlation method, resulting in an r value of -0.051 (FIG.10D) and to the date of diagnosis of ALS disease to the date of death using the Spearman correlation method, resulting in an r value of -0.181 (FIG.10E). Additionally, the ox-LDL expression level in the serum sample collected at the time of diagnosis from each of the 50 ALS patients was correlated to the duration of time between the date of onset of disease to the date of ALS disease diagnosis using the Spearman correlation method, resulting in an r value of 0.078 (FIG.10F). In contrast to 4-HNE and its correlative value to many aspects of survival, these data showed that ox-LDL alone does not correlate with NAI-1540949621v1 90Atty. Docket No.14678-018-228 1) mean ox-LDL serum expression levels and survival from the date of disease onset to the date of death, 2) mean ox-LDL serum expression levels and survival from the date of diagnosis to the date of death, 3) ox-LDL serum expression levels in the last sample collected prior to death and the duration of time between that last sample collection date to the date of death, 4) ox-LDL serum expression levels at diagnosis and survival from the date of onset to the date of death, 5) ox-LDL serum expression levels at diagnosis and survival from the date of diagnosis to the date of death, or 6) ox-LDL serum expression levels at diagnosis and the duration of time between the date of onset to the date of diagnosis.

[0285] To determine if ox-LDL serum expression levels correlate with disease progression, the rate of disease progression for each of the 50 ALS patients was determined as described above. Mean ox-LDL expression levels in the serum of each of the 50 patients with ALS studied longitudinally was correlated to the rate of disease progression determined for each of the 50 ALS patients using the Spearman correlation method which resulted in an r value of 0.158 (FIG. 11A). The ox-LDL expression level in the serum sample collected at the time of diagnosis from each of the 50 ALS patients was correlated to the rate of disease progression determined for each of the 50 ALS patients using the Spearman correlation method, resulting in an r value of -0.038 (FIG.11B). The ox-LDL expression level in the last serum sample collected from each of the 50 ALS patients prior to death was correlated to the rate of disease progression determined for each of the 50 ALS patients using the Spearman correlation method, resulting in an r value of 0.229 (FIG.11C). FIGS.11A-11C show that, unlike 4-HNE and rate of disease progression, there is no correlative value between 1) mean ox-LDL serum expression level and the rate of disease progression, 2) ox-LDL serum expression level at the time of diagnosis and the rate of disease progression, and 3) ox-LDL serum expression level at the date of last sample collection and the rate of disease progression. These data demonstrate that there is not a predictive value in ox-LDL serum expression levels over the course of ALS with rates of disease progression. 9. EQUIVALENTS

[0286] All patents and publications mentioned in this specification are incorporated herein by reference in their entireties. From the foregoing description, it will be apparent that variations and modifications can be made to the invention described herein to adopt it to various uses and conditions. Such embodiments are also within the scope of the following claims. NAI-1540949621v1 91

Claims

Atty. Docket No.14678-018-228 WHAT IS CLAIMED IS:

1. A method of predicting survival in a subject having or suspected of having amyotrophic lateral sclerosis (ALS), comprising: (a) determining a level of 4-hydroxynonenal (4-HNE) in a plasma or serum sample from the subject; and (b) diagnosing the subject as: (i) a long-term ALS survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short-term ALS survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE.

2. A method of predicting survival in a subject having or suspected of having amyotrophic lateral sclerosis (ALS), comprising: (a) obtaining or having obtained a plasma or serum sample from the subject; (b) determining a level of 4-hydroxynonenal (4-HNE) in the subject; and (c) diagnosing the subject as: (i) a long-term survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short-term survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE.

3. The method of claim 1 or 2, wherein the method further comprises: (d) administering: (i) a first therapeutic regimen to the subject if the subject is a long-term survivor, or (ii) a second therapeutic regimen to the subject if the subject is a short-term survivor.

4. The method of any one of claims 1 to 3, wherein determining the level of 4-HNE comprises an assay that uses a composition comprising a solid surface having an antibody or an antigen-binding portion thereof that binds to a 4-HNE epitope. NAI-1540949621v1 92Atty. Docket No.14678-018-228 5. The method of any one of claims 1 to 4, wherein the level of the 4-HNE in the sample equal to or lower than a reference range for 4-HNE correlates to an increased mean survival time for the subject.

6. The method of any one of claims 1 to 4, wherein the level of the 4-HNE in the sample greater than the reference range for 4-HNE correlates to a decreased mean survival time for the subject.

7. The method of any one of claims 1 to 4, wherein the level of the 4-HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about one point per month.

8. The method of any one of claims 1 to 4, wherein the level of the 4-HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about 0.7 points per month.

9. The method of any one of claims 1 to 4, wherein the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about one point per month.

10. The method of any one of claims 1 to 4, wherein the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about 1.2 points per month.

11. The method of any one of claims 1 to 10, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL.

12. The method of any one of claims 3 to 11, wherein the first therapeutic regimen and the second therapeutic regimen comprise an ALS therapy.

13. The method of claim 12, wherein the first therapeutic regimen and the second therapeutic regimen comprise the same ALS therapy.

14. The method of claim 12, wherein the first therapeutic regimen and the second therapeutic regimen comprise different ALS therapies.

15. The method of any one of claims 3 to 12, wherein the second therapeutic regimen comprises a palliative care therapeutic regimen.

16. The method of claim 13, wherein the second therapeutic regimen comprises a higher NAI-1540949621v1 93Atty. Docket No.14678-018-228 dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

17. The method of claim 13, wherein the second therapeutic regimen comprises a lower dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

18. The method of claim 13, wherein the second therapeutic regimen comprises administering a dosage of the ALS therapy with increased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

19. The method of claim 13, wherein the second therapeutic regimen comprises administering a dosage of the ALS therapy with decreased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

20. The method of claim 12, wherein the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, interleukin-2 (IL-2), abatacept (CTLA-4 Ig), or any combination thereof.

21. The method of claim 20, wherein the IL-2 is an IL-2 conjugate or an IL-2 mutein.

22. The method of claim 20, wherein the ALS therapy comprises an IL-2 / abatacept combination therapy.

23. The method of any one of claims 20 to 22, wherein the ALS therapy comprises a low dose of IL-2.

24. The method of claim 23, wherein the low dose of IL-2 comprises a dose of IL-2 less than about 5.0 ×106IU / m2 / day.

25. The method of any one of claims 20 to 24, wherein the IL-2 is a human recombinant IL-2.

26. The method of any one of claims 20 to 25, wherein the IL-2 is aldesleukin.

27. The method of claim 14, wherein the first ALS therapy or the second ALS therapy NAI-1540949621v1 94Atty. Docket No.14678-018-228 comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, IL-2, abatacept (CTLA-4 Ig), or any combination thereof.

28. The method of claim 15, wherein palliative care comprises management of one or more symptoms of ALS.

29. The method of claim 1, wherein the sample was obtained from the subject at ALS diagnosis.

30. A method of determining impact of an ALS therapy on survival of a subject having or suspected of having ALS, comprising: (a) determining a level of 4-hydroxynonenal (4-HNE) in a plasma or serum sample from the subject; (b) diagnosing the subject as: (i) a long-term ALS survivor if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) a short-term ALS survivor if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE; (c) administering an ALS therapy to the subject; (d) determining a level of 4-HNE in a plasma or serum sample from the subject following (c); and (e) comparing the level of 4-HNE in (a) to the 4-HNE level in (d), thereby determining the impact of the ALS therapy on survival of the subject.

31. The method of claim 30, wherein determining the level of 4-HNE comprises an assay that uses a composition comprising a solid surface having an antibody or an antigen-binding portion thereof that binds to a 4-HNE epitope.

32. The method of claim 30 or 31, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL.

33. The method of any one of claims 30 to 32, wherein the level of the 4-HNE in the sample equal to or lower than a reference range for 4-HNE correlates to an increased mean survival time for the subject. NAI-1540949621v1 95Atty. Docket No.14678-018-228 34. The method of any one of claims 30 to 32, wherein the level of the 4-HNE in the sample greater than the reference range for 4-HNE correlates to a decreased mean survival time for the subject.

35. The method of any one of claims 30 to 34, wherein the ALS therapy is predicted to improve survival of the subject when the 4-HNE level in (d) is equal to or less than the level of 4-HNE in (a).

36. The method of any one of claims 30 to 35, wherein the ALS therapy is predicted to have no effect or worsen survival of the subject when the 4-HNE level in (d) is higher than the level of 4-HNE in (a).

37. The method of either claim 35 or 36, wherein the method further comprises: (f) administering: (i) a continued dosage of the ALS therapy administered in (c) to the subject if the ALS therapy improves survival of the subject, or (ii) a second therapeutic regimen to the subject if the ALS therapy has no effect or worsens survival of the subject.

38. The method of any one of claims 30 to 37, wherein the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, interleukin-2 (IL-2), abatacept (CTLA-4 Ig), or any combination thereof.

39. The method of claim 38, wherein the IL-2 is an IL-2 conjugate or an IL-2 mutein.

40. The method of claim 38, wherein the ALS therapy comprises an IL-2 / abatacept combination therapy.

41. The method of any one of claims 38 to 40, wherein the ALS therapy comprises a low dose of IL-2.

42. The method of claim 41, wherein the low dose of IL-2 comprises a dose of IL-2 less than about 5.0 ×106IU / m2 / day.

43. The method of any one of claims 38 to 42, wherein the IL-2 is a human NAI-1540949621v1 96Atty. Docket No.14678-018-228 recombinant IL-2.

44. The method of any one of claims 38 to 43, wherein the IL-2 is aldesleukin.

45. The method of claim 37, wherein the second therapeutic regimen comprises a palliative care therapeutic regimen.

46. The method of claim 45, wherein palliative care comprises management of one or more symptoms of ALS.

47. The method of any one of claims 38 to 46, wherein the subject is naïve to ALS therapy.

48. The method of any one of claims 38 to 46, wherein the subject has previously received at least one ALS therapy.

49. A method of predicting the rate of ALS progression to disease endpoints in a subject having or suspected of having ALS, comprising: (a) obtaining or having obtained a plasma or serum sample from the subject; (b) determining a level of 4-hydroxynonenal (4-HNE) in a plasma or serum sample from the subject; and (c) diagnosing the subject as: (i) a slow ALS progressor if the level of the 4-HNE in the sample is equal to or lower than a reference range; or (ii) a rapid ALS progressor if the level of the 4-HNE in the sample is greater than the reference range, wherein the disease endpoints are death or a need for implementation of invasive respiratory measures.

50. The method of claim 49, wherein the method further comprises: (d) administering or withholding a first therapeutic regimen to the subject if the subject is a slow ALS progressor, and / or (e) administering a second therapeutic regimen to the subject if the subject is a rapid ALS progressor.

51. The method of claim 49 or 50, wherein determining the level of 4-HNE comprises an assay that uses a composition comprising a solid surface having an antibody or an antigen- NAI-1540949621v1 97Atty. Docket No.14678-018-228 binding portion thereof that binds to a 4-HNE epitope.

52. The method of any one of claims 49 to 51, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL.

53. The method of any one of claims 49 to 52, wherein the level of the 4-HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about one point per month.

54. The method of any one of claims 49 to 52, wherein the level of the 4-HNE in (c)(i) correlates with an ALSFRS-R rate of decline less than about 0.7 points per month.

55. The method of any one of claims 49 to 52, wherein the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about one point per month.

56. The method of any one of claims 49 to 52, wherein the level of the 4-HNE in (c)(ii) correlates with an ALSFRS-R rate of decline greater than about 1.2 points per month.

57. The method of any one of claims 50 to 56, wherein the first therapeutic regimen and the second therapeutic regimen comprise an ALS therapy.

58. The method of claim 57, wherein the first therapeutic regimen and the second therapeutic regimen comprise the same ALS therapy.

59. The method of claim 57, wherein the first therapeutic regimen and the second therapeutic regimen comprise different ALS therapies.

60. The method of any one of claims 50 to 57, wherein the second therapeutic regimen comprises a palliative care therapeutic regimen.

61. The method of claim 58, wherein the second therapeutic regimen comprises a higher dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

62. The method of claim 58, wherein the second therapeutic regimen comprises a lower dosage of the ALS therapy compared to the dosage of the ALS therapy administered in the first therapeutic regimen. NAI-1540949621v1 98Atty. Docket No.14678-018-228 63. The method of claim 58, wherein the second therapeutic regimen comprises administering a dosage of the ALS therapy with increased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

64. The method of claim 58, wherein the second therapeutic regimen comprises administering a dosage of the ALS therapy with decreased frequency compared to the dosage of the ALS therapy administered in the first therapeutic regimen.

65. The method of claim 57, wherein the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, interleukin-2 (IL-2), abatacept (CTLA-4 Ig), or any combination thereof.

66. The method of claim 65, wherein the IL-2 is an IL-2 conjugate or an IL-2 mutein.

67. The method of claim 65, wherein the ALS therapy comprises an IL-2 / abatacept combination therapy.

68. The method of any one of claims 65 to 67, wherein the ALS therapy comprises a low dose of IL-2.

69. The method of claim 68, wherein the low dose of IL-2 comprises a dose of IL-2 less than about 5.0 ×106IU / m2 / day.

70. The method of any one of claims 65 to 69, wherein the IL-2 is a human recombinant IL-2.

71. The method of any one of claims 65 to 70, wherein the IL-2 is aldesleukin.

72. The method of claim 59, wherein the first ALS therapy or the second ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, IL-2, abatacept (CTLA-4 Ig), or any combination thereof.

73. The method of claim 60, wherein palliative care comprises management of one or more symptoms of ALS.

74. A method of treating ALS, comprising administering to a subject predicted to be a short- NAI-1540949621v1 99Atty. Docket No.14678-018-228 term ALS survivor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a short-term ALS survivor in that serum or plasma collected from the subject comprises a 4-HNE level greater than a 4-HNE reference range.

75. A method of treating ALS, comprising administering to a subject predicted to be a long- term ALS survivor a therapeutically effective amount of an ALS therapy, wherein the subject is predicted to be a long-term ALS survivor in that serum or plasma collected from the subject comprises a 4-HNE level equal to or lower than a 4-HNE reference range.

76. The method of claim 74 or 75, wherein determining a 4-HNE level comprises an assay comprising a solid surface having an antibody or an antigen-binding portion thereof that binds to a 4-HNE epitope.

77. The method of any one of claims 74 to 76, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL.

78. The method of any one of claims 74 to 77, wherein the ALS therapy comprises a Treg infusion, sodium phenylbutyrate / taurursodiol, edaravone, riluzole, IL-2, abatacept (CTLA-4 Ig), or any combination thereof.

79. A kit for detecting and quantifying the level of 4-hydroxynonenal (4-HNE) in a serum or plasma sample obtained from a subject having or suspected of having ALS, wherein the kit comprises a solid surface having an antibody or a fragment thereof comprising a binding epitope for 4-HNE.

80. The kit of claim 79, further comprising at least one reagent for detecting formation of a 4- HNE-antibody complex.

81. The kit of claim 79 or claim 80 for use in predicting a survival outcome in the subject having or suspected of having ALS.

82. The kit of any one of claims 79 to 81 for use in monitoring changes in a survival outcome during the course of or after completing an ALS therapy regimen.

83. A method of predicting clinical benefit of an ALS therapy comprising: NAI-1540949621v1 100Atty. Docket No.14678-018-228 (a) administering the ALS therapy to a subject; (b) collecting a plasma or serum sample from the subject after administering the ALS therapy; (c) determining the level of 4-HNE in the sample collected from the subject, wherein the level of 4-HNE in the sample acts as a surrogate endpoint; (d) predicting the clinical benefit of the ALS therapy as: (i) favorable if the level of the 4-HNE in the sample is equal to or lower than a reference range for 4-HNE; or (ii) unfavorable if the level of the 4-HNE in the sample is greater than the reference range for 4-HNE.

84. The method of claim 83, wherein a favorable clinical benefit of the ALS therapy is correlated with slow disease progression.

85. The method of claim 83, wherein an unfavorable clinical benefit of the ALS therapy is correlated with rapid disease progression.

86. The method of claim 83 or 85, wherein an unfavorable clinical benefit of the ALS therapy is predictive of death.

87. The method of claim 83, wherein the subject is not administered the ALS therapy after the level of the 4-HNE in the sample is determined to be greater than the reference range for 4- HNE.

88. The method of any one of claims 83 to 87, wherein the reference range for 4-HNE is about 0.5 µg / mL to about 12 µg / mL.

89. The method of any one of claims 83 to 88, wherein the clinical benefit of the ALS therapy is unknown prior to administration.

90. The method of any one of claims 83 to 89, wherein use of the level of 4-HNE in the sample as surrogate endpoint can predict clinical benefit in a clinical trial of at least one investigational therapy for ALS. NAI-1540949621v1 101

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